Treatment of heart disease by inhibtion of the action of muscle A-kinase anchoring protein (mAKAP)

ABSTRACT

The present invention provides a method of protecting the heart from damage, by administering to a patient at risk of such damage, a pharmaceutically effective amount of a composition which inhibits the interaction of RSK3 and mAKAPβ, or the expression or activity of one or both of those molecules. This composition is preferably in the form of an siRNA construct, more preferably an shRNA construct, which inhibits the expression of mAKAPβ.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/529,224, filed Jul. 6, 2017, which is hereby incorporated byreference in its entirety into the present application.

This application also incorporates by reference in their entireties U.S.patent application Ser. No. 14/821,082, filed Aug. 7, 2015, now U.S.Pat. No. 9,937,228, issued n Apr. 10, 2018, U.S. patent application Ser.No. 14/213,583, filed on Mar. 14, 2014, now U.S. Pat. No. 9,132,174,issued on Sep. 15, 2015, and U.S. Provisional Application No.61/798,268, filed Mar. 15, 2013.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with Government support under contract RO1 HL075398 awarded by the National Institutes of Health. The Government hascertain rights in this invention.

REFERENCE TO SEQUENCE LISTING, COMPUTER PROGRAM, OR COMPACT DISK

The present application includes a Sequence Listing filed in electronicformat. The Sequence Listing is entitled “4175-103US_ST25.txt” createdon Sep. 25, 2018, and is 76,000 bytes in size. The information in theelectronic format of the Sequence Listing is part of the presentapplication and is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The heart is capable of undergoing hypertrophic growth in response to avariety of stimuli. Hypertrophic growth may occur as the result ofphysical training such as running or swimming. However, it also occursas the result of injury or in many forms of heart disease. Hypertrophyis the primary mechanism by which the heart reduces stress on theventricular wall. When the growth is not accompanied by a concomitantincrease in chamber size, this is called concentric hypertrophy.Hypertrophy occurs as the result of an increase in protein synthesis andin the size and organization of sarcomeres within individual myocytes.For a more thorough review of cardiac remodeling and hypertrophy, seeKehat (2010) and Hill (2008), each herein incorporated by reference intheir entirety. The prevailing view is that cardiac hypertrophy plays amajor role in heart failure. Traditional routes of treating heartfailure include afterload reduction, blockage of beta-adrenergicreceptors (β-ARs) and use of mechanical support devices in afflictedpatients. However, the art is in need of additional mechanisms ofpreventing or treating cardiac hypertrophy.

AKAPs and Cardiac Remodeling

Ventricular myocyte hypertrophy is the primary compensatory mechanismwhereby the myocardium reduces ventricular wall tension when submittedto stress because of myocardial infarction, hypertension, and congenitalheart disease or neurohumoral activation. It is associated with anonmitotic growth of cardiomyocytes, increased myofibrillarorganization, and upregulation of specific subsets of “fetal” genes thatare normally expressed during embryonic life (Frey 2004, Hill 2008). Theconcomitant aberrant cardiac contractility, Ca²⁺ handling, andmyocardial energetics are associated with maladaptive changes thatinclude interstitial fibrosis and cardiomyocyte death and increase therisk of developing heart failure and malignant arrhythmia (Cappola 2008,Hill 2008). Increased in prevalence by risk factors such as smoking andobesity, heart failure is a syndrome that affects about six millionAmericans and has an annual incidence of 1% of senior citizens (Roger2011). Since the five-year survival rate after diagnosis is still verypoor (lower than 50%), many efforts have been made during the last yearsto define the molecular mechanisms involved in this pathologicalprocess.

Cardiac hypertrophy can be induced by a variety of neuro-humoral,paracrine, and autocrine stimuli, which activate several receptorfamilies including G protein-coupled receptors, cytokine receptors, andgrowth factor tyrosine kinase receptors (Brown 2006, Frey 2004). In thiscontext, it is becoming increasingly clear that A-kinase anchoringproteins (AKAPs) can assemble multiprotein complexes that integratehypertrophic pathways emanating from these receptors. In particular,recent studies have now identified anchoring proteins including mAKAPand AKAP-Lbc and D-AKAP1 that serve as scaffold proteins and play acentral role in organizing and modulating hypertrophic pathwaysactivated by stress signals.

As the organizers of “nodes” in the intracellular signaling network,scaffold proteins are of interest as potential therapeutic targets(Negro 2008). In cells, scaffold proteins can organize multimolecularcomplexes called “signalosomes,” constituting an important mechanismresponsible for specificity and efficacy in intracellular signaltransduction (Scott and Pawson 2009). Firstly, many signaling enzymeshave broad substrate specificity. Scaffold proteins can co-localizethese pleiotropic enzymes with individual substrates, selectivelyenhancing the catalysis of substrates and providing a degree ofspecificity not intrinsic to the enzyme's active site (Scott and Pawson2009). Secondly, some signaling enzymes are low in abundance. Scaffoldproteins can co-localize a rare enzyme with its substrate, makingsignaling kinetically favorable. Thirdly, since many scaffolds aremultivalent, scaffold binding can orchestrate the co-regulation bymultiple enzymes of individual substrate effectors. Muscle A-kinaseanchoring protein (mAKAP, a.k.a. AKAP6) is a large scaffold expressed incardiac and skeletal myocytes and neurons that binds both signalingenzymes such as protein kinase A (PKA) and the Ca²⁺/calmodulin-dependentphosphatase Calcineurin (CaN) that have broad substrate specificity andsignaling enzymes such as p90 ribosomal S6 kinase 3 (RSK3) that isremarkably low in abundance (FIGS. 1 and 7) (Kapiloff 1999, Michel 2005,Pare 2005, Wang 2015). mAKAPβ is the alternatively-spliced isoformexpressed in myocytes, in which cells it is localized to the outernuclear membrane by binding the integral membrane protein nesprin-1α(Pare 2005).

mAKAPβ “signalosomes” regulate cardiac remodeling by multiple mechanisms(FIGS. 1 and 7) (Passariello 2015). By binding a diverse set ofsignaling molecules, some constitutively and some in response tostress-related stimuli, mAKAPβ dynamically organizes multiple signalingmodules that transduce cAMP, mitogen-activated protein kinase (MAPK),calcium, phosphoinositide, and hypoxic stress signals (Marx 2000,Kapiloff 2001, Dodge-Kafka 2005, Wong 2008, Kapiloff 2009, Li 2010,Vargas 2012, Kritzer 2014). mAKAP was originally identified by itsbinding to PKA, and mAKAPβ signalosomes contain all of the requiredmachinery for cAMP synthesis, degradation and function, includingadenylyl cyclase 5, type 4D3 phosphodiesterase (PDE4D3), and the cAMPtargets type IIα PKA and exchange protein activated by cAMP-1 (Epac1)(Kapiloff 1999, Dodge 2001, Dodge-Kafka 2005, Kapiloff 2009). Notably,all of these signaling enzymes have been implicated in myocyteremodeling, and binding of PKA to mAKAPβ has been shown to be importantfor myocyte hypertrophy (Okumura 2003, Dodge-Kafka 2005, Lehnart 2005,Pare 2005). In addition, mAKAPβ binds a wide variety of other proteinsimportant for myocyte stress responses: MEK5 and ERK5 MAP-kinases,3-phosphoinositide-dependent protein kinase-1 (PDK1), RSK3,phospholipase Cε (PLCε), protein kinase Cε (PKCε), protein kinase D(PKD, PKCμ), the protein phosphatases CaN (Aβ isoform) and PP2A, thetype 2 ryanodine receptor (RyR2), the sodium/calcium exchanger NCX1,ubiquitin E3-ligases involved in HIF1α regulation, and myopodin (Marx2000, Kapiloff 2001, Schulze 2003, Dodge-Kafka 2005, Michel 2005, Pare2005, Pare 2005, Dodge-Kafka and Kapiloff 2006, Faul 2007, Wong 2008,Kapiloff 2009, Zhang 2011, Vargas 2012, Li, Zhang 2013). Bound tomAKAPβ, these signaling molecules co-regulate the transcription factorshypoxia-inducible factor 1α (HIF1α), myocyte enhancer factor-2 (MEF2),and nuclear factor of activated T-cell (NFATc), as well as type IIahistone deacetylases (HDAC4/5) (Wong 2008, Li 2010, Li 2013, Kritzer2014).

Consistent with its role as a scaffold protein for stress-relatedsignaling molecules in the cardiac myocyte, depletion of mAKAPβ in ratneonatal ventricular myocytes in vitro inhibited hypertrophy induced byα-adrenergic, β-adrenergic, endothelin-1, angiotensin II, and leucineinhibitor factor/gp130 receptor signaling (Dodge-Kafka 2005, Pare 2005,Zhang 2011, Guo 2015). In vivo, along with attenuating hypertrophyinduced by short-term pressure overload and chronic β-adrenergicstimulation, mAKAP gene targeting in the mouse inhibited the developmentof heart failure following long-term pressure overload, conferring asurvival benefit (Kritzer 2014). Specifically, mAKAP gene deletion inthe mAKAP^(fl/fl); Tg(Myh6-cre/Esr1*), tamoxifen-inducible, conditionalknock-out mouse reduced left ventricular hypertrophy, while greatlyinhibiting myocyte apoptosis, and interstitial fibrosis, left atrialhypertrophy, and pulmonary edema (wet lung weight) due to transverseaortic constriction for 16 weeks (Kritzer 2014).

mAKAP gene targeting is also beneficial following myocardial infarction.Permanent ligation of the left anterior descending coronary artery (LAD)in the mouse results in myocardial infarction, including extensivemyocyte death, scar formation, and subsequent left ventricular (LV)remodeling. Four weeks following LAD ligation, mAKAP conditionalknock-out mouse had preserved LV dimensions and function when tocompared to infarcted control cohorts. mAKAP conditional knock-out micehad preserved LV ejection fraction and indexed atrial weight compared tocontrols, while displaying a remarkable decrease in infarct size.

In cardiomyocytes, mAKAPβ is localized to the nuclear envelope throughan interaction with nesprin-1α (Pare 2007). mAKAPβ assembles a largesignaling complex that integrates hypertrophic signals initiated byα1-adrenergic receptors (α1-ARs) and β-ARs, endothelin-1 receptors, andgp130/leukemia inhibitor factor receptors (FIG. 46A of of U.S. Pat. No.9,132,174) (Dodge-Kafka 2005, Pare 2005). Over the last few years, themolecular mechanisms as well as the signaling pathways whereby mAKAPβmediates cardiomyocyte hypertrophy have been extensively investigated.It is now demonstrated that mAKAPβ can recruit the phosphatasecalcineurin Aβ (CaNAβ) as well as the hypertrophic transcription factornuclear factor of activated T cells c3 (NFATc3) (Li 2010). In responseto adrenergic receptor activation, anchored CaNAβ dephosphorylates andactivates NFATc3, which promotes the transcription of hypertrophic genes(FIG. 46A of U.S. Pat. No. 9,132,174) (Li 2010). The molecularmechanisms controlling the activation of the pool of CaNAβ bound to themAKAPβ complex are currently not completely understood but seem torequire mobilization of local Ca²⁺ stores. In this context, it has beenshown that mAKAP favors PKA-induced phosphorylation of RyR2 (Kapiloff2001), which, through the modulation of perinuclear Ca²⁺ release, couldactivate CaNAβ (FIG. 46A of U.S. Pat. No. 9,132,174). In line with thishypothesis, the deletion of the PKA anchoring domain from mAKAPβ hasbeen shown to suppress the mAKAP-mediated hypertrophic response (Pare2005). On the other hand, recent findings indicate that mAKAPβ alsobinds phospholipase Cε(PLCε) and that disruption of endogenousmAKAPβ-PLCε complexes in rat neonatal ventricular myocytes inhibitsendothelin 1-induced hypertrophy (Zhang 2011). This suggests that theanchoring of PLCε to the nuclear envelope by mAKAPβ controlshypertrophic remodeling. Therefore, it is also plausible that at thenuclear envelope, PLCε might promote the generation of inositol1,4,5-trisphosphate, which through the mobilization of local Ca²⁺stores, might promote the activation of CaNAβ and NFATc3 bound to mAKAPβ(FIG. 46 of U.S. Pat. No. 9,132,174).

In cardiomyocytes, the dynamics of PKA activation within the mAKAPcomplex are tightly regulated by AC5 (Kapiloff 2009) and the PDE4D3(Dodge-Kafka 2005, Dodge 2001) that are directly bound to the anchoringprotein. The mAKAP-bound AC5 and upstream β-AR may be localized withintransverse tubules adjacent to the nuclear envelope (Escobar 2011). Inresponse to elevated cAMP levels, mAKAP-bound PKA phosphorylates bothAC5 and PDE4D3 (Dodge-Kafka 2005, Dodge 2001, Kapiloff 2009). Thisinduces AC5 deactivation and PDE4D3 activation, which locally decreasescAMP concentration and induces deactivation of anchored PKA (FIG. 46A ofU.S. Pat. No. 9,132,174). Dephosphorylation of PDE4D3 is mediated by thephosphatase PP2A that is also associated with mAKAPβ (FIG. 46A of U.S.Pat. No. 9,132,174) (Dodge-Kafka 2010). Collectively, these findingssuggest that the mAKAP complex generates cyclic pulses of PKA activity,a hypothesis that was supported experimentally by live cell imagingstudies (Dodge-Kafka 2005).

AKAPs and Hypoxia

Myocardial oxygen levels need to be maintained within narrow levels tosustain cardiac function. During ischemic insult, in response toconditions of reduced oxygen supply (termed hypoxia), cardiomyocytesmobilize hypoxia-inducible factor 1α (HIF-1α), a transcription factorthat promotes a wide range of cellular responses necessary to adapt toreduced oxygen (Semenza 2007). Transcriptional responses activated byHIF-1α control cell survival, oxygen transport, energy metabolism, andangiogenesis (Semenza 2007). Under normoxic conditions, HIF-1α ishydroxylated on two specific proline residues by the prolyl hydroxylasedomain proteins (PHDs) and subsequently recognized and ubiquitinated bythe von Hippel-Lindau protein (Jaakkola 2001, Maxwell 1999).Ubiquitinated HIF-1α is targeted to the proteasome for degradation. Onthe other hand, when oxygen concentration falls, the enzymatic activityof PHD proteins is inhibited. Moreover, PHD proteins are ubiquitinatedby an E3 ligase named “seven in absentia homolog 2 (Siah2)” and targetedfor proteasomal degradation (Nakayama 2004). This inhibits HIF-1αdegradation and allows the protein to accumulate in the nucleus where itpromotes gene transcription required for the adaptive re-sponse tohypoxia. In line with this finding, the delivery of exogenous HIF-1αimproves heart function after myocardial infarction (Shyu 2002), whereascardiac overexpression of HIF-1α reduces infarct size and favors theformation of capillaries (Kido 2005).

Recent findings indicate that mAKAP assembles a signaling complexcontaining HIF-1α, PHD, von Hippel-Lindau protein, and Siah2 (Wong2008). This positions HIF-1α in proximity of its upstream regulators aswell as to its site of action inside the nucleus. In this configuration,under normoxic conditions, negative regulators associated with the mAKAPcomplex favor HIF-1α degradation (Wong 2008). On the other hand, duringhypoxia, the activation of Siah2 within the mAKAP complex promotesHIF-1α stabilization, allowing the transcription factor to inducetranscription (Wong 2008). Therefore, mAKAP assembles a macromolecularcomplex that can favor degradation or stabilization of HIF-1α incardiomyocytes in response to variations of oxygen concentrations. Inthis context, mAKAP could play an important role in cardiomyocyteprotection during cardiac ischemia, when coronary blood flow is reducedor interrupted. By coordinating the molecular pathways that controlHIF-1α stabilization in cardiomyocytes, mAKAP might favorHIF-1α-mediated transcriptional responses, controlling the induction ofglycolysis (which maximizes ATP production under hypoxic conditions),the efficiency of mitochondrial respiration, and cell survival duringischemia (Semenza 2009).

RSKs and Cardiac Remodeling

Myofibrillar assembly driving nonmitotic growth of the cardiac myocyteis the major response of the heart to increased workload (Kehay 2010).Although myocyte hypertrophy per se may be compensatory, in diseasessuch as hypertension and myocardial infarction, activation of thehypertrophic signaling network also results in altered gene expression(“fetal”) and increased cellular apoptosis and interstitial fibrosis,such that left ventricular hypertrophy is a major risk factor for heartfailure. Current therapy for pathologic hypertrophy is generally limitedto the broad downregulation of signaling pathways through the inhibitionof upstream cell membrane receptors and ion channels (McKinsey 2007).Novel drug targets may be revealed through the identification ofsignaling enzymes that regulate distinct pathways within thehypertrophic signaling network because of isoform specificity orassociation with unique multimolecular signaling complexes.

p90 ribosomal S6 kinases (RSK) are pleiotropic extracellularsignal-regulated kinase (ERK) effectors with activity that is increasedin myocytes by most hypertrophic stimuli (Anjum 2008, Sadoshima 2005,Kodama 2000). In addition, increased RSK activity has been detected inexplanted hearts from patients with end-stage dilated cardiomyopathy(Takeishi 2002). There are 4 mammalian RSK family members that areubiquitously expressed and that overlap in substrate specificity (Anjum2008). RSKs are unusual in that they contain 2 catalytic domains,N-terminal kinase domain and C-terminal kinase domain (FIG. 4A of U.S.Pat. No. 9,132,174, Anjum 2008). The N-terminal kinase domainphosphorylates RSK substrates and is activated by sequentialphosphorylation of the C-terminal kinase domain and N-terminal kinasedomain by ERK (ERK1, ERK2, or ERK5) and 3′-phosphoinositide-dependentkinase 1 (PDK1), respectively (Anjum 2008).

By binding scaffold proteins, RSKs may be differentially localizedwithin subcellular compartments, conferring isoform-specific signalinglike when bound to the scaffold protein muscle A-kinase anchoringprotein (mAKAP) (Michael 2005). PDK1 activation of RSK was enhanced byco-expression with the mAKAP scaffold in a recombinant system. Incardiac myocytes, mAKAPβ (the alternatively spliced form expressed inmuscle cells) organizes signalosomes that transduce cAMP,mitogen-activated protein kinase, Ca²⁺, and hypoxic signaling by bindinga diverse set of enzymes, ion channels, and transcription factors(Kritzer 2012).

In the United States, heart failure affects 5.7 million people, and eachyear 915,000 new cases are diagnosed (Writing Group 2016). Theprevalence and incidence of heart failure are increasing, mainly becauseof increasing life span, but also because of the increased prevalence ofrisk factors (hypertension, diabetes, dyslipidemia, and obesity) andimproved survival rates from other types of cardiovascular disease(myocardial infarction [MI] and arrhythmias) (Heidenreich 2013).First-line therapy for patients with heart failure includesangiotensin-converting enzyme (ACE) inhibitors and β-adrenergic receptorblockers (β-blockers) that can improve the survival and quality of lifeof such patients, as well as reduce mortality for those with leftventricular dysfunction (Consensus 1987). Subsequent or alternativetherapies include aldosterone and angiotensin II receptor blockers,neprilysin inhibitors, loop and thiazide diuretics, vasodilators, andI_(f) current blockers, as well as device-based therapies (Ponikowski2016). Nevertheless, the 5-year mortality for symptomatic heart failureremains 50%, including >40% mortality for those post-MI (Heidenreich2013, Gerber 2016). There is a clear need to develop new effectivetherapies to treat patients with heart failure, as well as to preventits development in the context of other cardiovascular diseases suchcoronary artery disease, hypertension, and valvular disease.

SUMMARY OF THE INVENTION

The following brief summary is not intended to include all features andaspects of the present invention, nor does it imply that the inventionmust include all features and aspects discussed in this summary.

The present inventors have discovered methods of treating cardiacpathological processes by inhibiting the signaling properties ofindividual mAKAP signaling complexes using drugs that target uniqueprotein-protein interactions. Such a therapeutic strategy offers anadvantage over classical therapeutic approaches because it allows theselective inhibition of defined cellular responses.

In particular, the present inventors have found that disruptingmAKAP-mediated protein-protein interactions can be used to inhibit theability of mAKAP to coordinate the activation of enzymes that play acentral role in activating key transcription factors that initiate theremodeling process leading to cardiac hypertrophy.

Specifically, the inventors have discovered that inhibiting the bindinginteraction between type 3 ribosomal S6 kinase (RSK3) and mAKAPβ canprotect the heart from damage caused by various physical stresses, forexample pressure overload and prolonged exposure to high levels ofcatecholamines.

Thus, the present invention comprises, in certain aspects a method forprotecting the heart from damage, by administering to a patient at riskof such damage, a pharmaceutically effective amount of a compositionwhich inhibits the interaction of RSK3 and mAKAPβ.

The invention also relates to a method of treating heart disease, byadministering to a patient a pharmaceutically effective amount of acomposition which inhibits the interaction of RSK3 and mAKAPβ.

The invention also relates to compositions which inhibit the interactionof RSK3 and mAKAPβ.

In still other embodiments, the inhibitors include any molecule thatinhibits the expression or activity of of RSK3 and mAKAPβ.

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. Model for RSK3 signaling. MAP-Kinase signaling induced byα1-adrenergic receptor (α1-AR) stimulation and potentially otherupstream signals activates anchored RSK3 in conjunction with PDK1 atanchored sites, including at perinuclear mAKAPβ signalosomes. Targetsfor RSK3 may include cytosolic and nuclear proteins, especially thoseinvolved in the regulation of hypertrophic gene expression.

FIG. 2. Shows the amino acid sequence of human RSK3 (SEQ ID NO: 1).

FIG. 3. Shows the amino acid sequence of rat mAKAP (SEQ ID NO: 2). Notethat within this document, references to mAKAP sequences, whetherlabelled “mAKAPβ” or “mAKAP” are according to the numbering for themAKAPα alternatively-spliced form which contains within the entirety ofmAKAPβ and is identical to the originally published mAKAP sequence asshown in this figure (Kapiloff 1999, Michel 2005). “mAKAP” is alsoreferred to as “AKAP6” in reference databases and the literature.

FIG. 4 shows cDNA cloning, in vitro translation, and detection ofendogenous RSK3 by immunoblotting. FIG. 4(a) shows the completenucleotide (SEQ ID NO:3) and deduced amino acid (SEQ ID NO:4) sequenceof human RSK3. (Zhao (1995)). The sequence was derived from afull-length cDNA clone. The deduced RSK3 protein sequence is indicatedin the one-letter amino acid code beginning at the first methionineresidue preceding the 733-codon open reading frame and terminating atthe asterisk. Highly conserved amino acid residues among the knownprotein kinases are shown in boldface type. The unique N-terminal regionof RSK3 (which bears no homology to RSK1 or RSK2) is underlined; theputative bipartite nuclear targeting motif is indicated by parentheses.An in-frame stop codon upstream of the first methionine is indicated(***). This nucleotide sequence was submitted to the EMBL-GenBank datalibrary and assigned accession number X85106. FIG. 4(b) shows in vitrotranslation of RSK3. In vitro transcripts were generated with T7polymerase from the vector alone (lane 1) or from the vector with anRSK3 insert by using T7 polymerase (lane 2, sense oriented) or Sp6polymerase (lane 3, antisense oriented). Subsequent in vitro translationwas performed with rabbit reticulocyte lysate in the presence of[³⁵S]methionine. Proteins were then resolved by SDSPAGE (10%polyacrylamide) followed by autoradiography. FIG. 4(c) showsimmunoblotting with RSK3-specific antiserum. Antiserum N-67 was raisedagainst a peptide (KFA VRRFFSVYLRR) derived from the unique N-terminalregion of RSK3 (residues 7 to 20 of SEQ ID NO:1). In this example,proteins derived from human skin fibroblasts were separated by SDS-PAGEfollowed by Western immunoblotting. Blots were probed with preimmuneserum or N-67. A band of 83 kDa was detected when N-67 was used. (Zhao(1995)).

FIG. 5. Shows the nucleotide sequence of human RSK3 (SEQ ID NO: 5).

FIG. 6. Shows the nucleotide sequence of rat mAKAP (SEQ ID NO: 6).

FIG. 7. mAKAPβ Signalosomes Drive Pathological Remodeling. ERK pathwaysare activated by stress signaling. Anchored RSK3 is activated by ERK and3′-phosphoinositide-dependent protein kinase 1 (PDK1)-catalyzedphosphorylation (Martinez 2015). Calcineurin (CaN) bound to mAKAPβ isimportant for the activation of MEF2D and NFATc transcription factors(Li 2010, Li 2013). mAKAP-anchored protein kinase D (PKD)dephosphorylates type IIa histone deacetylases, de-repressing geneexpression (Zhang 2013, Kritzer 2014). Together these pathways promotepathological gene expression.

FIG. 8. Sequence of human mAKAP (AKAP6) mRNA (SEQ ID NO:11)—ref seqXM_017021808.1 with shRNA sequences (#1-3) marked. Numbering is fornucleotide sequence. Encoded animo acids are indicated above.

FIG. 9. Map of pscA-TnT-mAKAP shrna (#3) plasmid.

FIG. 10. Nucleotide sequence of pscA-TnT-mAKAP shrna (#3) plasmid (SEQID NO:12) with key features and some restriction enzymes sitesindicated.

FIG. 11. Design of a scAAV-mAKAP shRNA biologic drug. A. mAKAP mRNAsequences form human (SEQ ID NO:13), swine (SEQ ID NO:14), mouse (SEQ IDNO:15) and rat (SEQ ID NO:16). The boxed sequence is shRNA target #3. B.AAV Design. A truncated right ITR confers self-complementarity. cTnT isthe cardiac myocyte-specific promoter. C. Western blot of total heartextracts for mAKAPβ 3 weeks after injection into adult mice with 5×10¹¹vg IV scAAV-mAKAP shRNA #3 or scAAV-control shRNA (containing a randomsequence). NI—non-injected control.

FIG. 12. Inhibition of mAKAPβ post-MI Ameliorates Left Ventricular (LV)Systolic Dysfunction in C57BL/6 mice. A. Experimental design. Myocardialinfarction (MI) was induced by permanent ligation of the left anteriorcoronary artery survival surgery. Sham-operated mice underwent all butcoronary artery ligation. AAV IV refers to tail vein injection of AAVbiologic. B. Administration of a single intravenous injection ofscAAV-mAKAP-shRNA #3 (shR-mAKAP-MI) 3 days post-MI resulted inamelioration of systolic dysfunction as evidenced by normalization ofejection fraction (EF) from day 2 to weeks 2, 4 and 8 post-MI. Incontrast, mice treated with scAAV-control-shRNA (shRNA-Ctrl-MI)displayed a progressive deterioration of EF after ischemic injury. At 8weeks post MI, EF in shRNA-mAKAP-MI was not significantly different thaneither mAKAP- and control-shRNA-injected Sham-operated animals (44.3±3.3vs. 54.6±1.7 & 54.5±2.0%), while significantly greater than that forcontrol-shRNA-MI animals (18.0±4.3%, P<0.0001).

FIG. 13. Post-mortem gravimetrics for mice studied in FIG. 12.scAAV-mAKAP-shRNA #3 treatment improved overall heart weight,biventricular weight, atrial weight, and wet lung weight.

DETAILED DESCRIPTION OF THE INVENTION

As discussed above, AKAP-based signaling complexes play a central rolein regulating physiological and pathological cardiac events. As such,the present inventors have examined inhibiting the signaling propertiesof individual AKAP signaling complexes using drugs that target uniqueprotein-protein interactions as an approach for limiting cardiacpathological processes. Such a therapeutic strategy offers an advantageover classical therapeutic approaches since it allows the selectiveinhibition of defined cellular responses.

Anchoring proteins including mAKAP are therapeutic targets for thetreatment of cardiac hypertrophy and heart failure. In particular, thepresent inventors have found that disrupting AKAP-mediatedprotein-protein interactions can be used to inhibit the ability of mAKAPto coordinate the activation of enzymes that play a central role inactivating key transcription factors that initiate the remodelingprocess leading to cardiac hypertrophy.

In particular, the inventors have found that type 3 ribosomal S6 kinase(RSK3) binds mAKAPβ directly via the unique N-terminal domain of RSK3,defining a novel enzyme-scaffold interaction. The inventors have foundthat anchored RSK3 regulates concentric cardiac myocyte growth,revealing an isoform-specific target for therapeutic intervention inpathologic cardiac hypertrophy. Delivery of such peptides that mightinhibit RSK3-mAKAPβ interaction can be enhanced by the use ofcell-penetrating sequences such as the transactivator of transcriptionpeptide and polyarginine tails, or conjugation with lipid-derived groupssuch as stearate. Stability may also be enhanced by the use ofpeptidomimetics [i.e., peptides with structural modifications in theoriginal sequence giving protection against exo- and endoproteaseswithout affecting the structural and functional properties of thepeptide. Alternatively, as shown in FIG. 41 of U.S. Pat. No. 9,132,174,peptides can be delivered by intracellular expression via viral-basedgene therapy vectors.

The inventors have also found that small molecule disruptors can be usedto target specific interaction within AKAP-based complexes. Smallmolecule disruptors can be identified by combining rational design andscreening approaches. Such compounds can be designed to target-specificbinding surfaces on AKAPs, to disrupt the interaction between AKAPs andPKA in cardiomyocytes and to enhance the contractility of intact heartsfor the treatment of chronic heart failure.

The present invention relates to methods of treating any cardiaccondition, which is initiated through the interaction of RSK3 andmAKAPβ. Such cardiac dysfunction can result in signs and symptoms suchas shortness of breath and fatigue, and can have various causes,including, but not limited to hypertension, coronary artery disease,myocardial infarction, valvular disease, primary cardiomyopathy,congenital heart disease, arrhythmia, pulmonary disease, diabetes,anemia, hyperthyroidism and other systemic diseases.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, “Molecular Cloning:A Laboratory Manual” (4th Ed., 2012); “Current Protocols in MolecularBiology” Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: ALaboratory Handbook” Volumes I-III [J. E. Celis, 3rd ed. (2005))];“Current Protocols in Immunology” Volumes I-III [Coligan, J. E., ed.(2005)]; “Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “NucleicAcid Hybridization” [B. D. Hames & S. J. Higgins eds. (1985)];“Transcription And Translation” [B. D. Hames & S. J. Higgins, eds.(1984)]; “Animal Cell Culture” [R. I. Freshney, ed. (1986)];“Immobilized Cells And Enzymes” [IRL Press, (1986)]; B. Perbal, “APractical Guide To Molecular Cloning” (1984); C. Machida, “Viral Vectorsfor Gene Therapy: Methods and Protocols” (2010); J. Reidhaar-Olson andC. Rondinone, “Therapeutic Applications of RNAi: Methods and Protocols”(2009).

The following definitions and acronyms are used herein:

ANF atrial natriuretic factor CTKD C-terminal kinase domain ERKextracellular signal-regulated kinase FBS fetal bovine serum GFP greenfluorescent protein Iso isoproterenol LIF leukemia inhibitory factormAKAP muscle A-kinase anchoring protein MI myocardial infarction NTKDN-terminal kinase domain PDK1 3′phosphoinositide-dependent kinase 1 PEphenylephrine RBD RSK binding domain RSK p90 ribosomal S6 kinase siRNAsmall interfering RNA oligonucleotide shRNA short hairpin RNA TACtransverse aortic constriction

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. Generally, nomenclatures utilized inconnection with, and techniques of, cell and molecular biology andchemistry are those well known and commonly used in the art. Certainexperimental techniques, not specifically defined, are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification. For purposes of theclarity, following terms are defined below.

The present invention recognizes that the interaction of RSK3 and mAKAPβmediates various intracellular signals and pathways which lead tocardiac myocyte hypertrophy and/or dysfunction. As such, the presentinventors have discovered various methods of inhibiting that interactionin order to prevent and/or treat cardiac myocyte hypertrophy and/ordysfunction.

Thus, the present invention includes a method for protecting the heartfrom damage, by administering to a patient at risk of such damage, apharmaceutically effective amount of a composition, which inhibits theinteraction of RSK3 and mAKAPβ. It should be appreciated that “apharmaceutically effective amount” can be empirically determined basedupon the method of delivery, and will vary according to the method ofdelivery.

The invention also relates to a method of treating heart disease, byadministering to a patient a pharmaceutically effective amount of acomposition, which inhibits the interaction of RSK3 and mAKAPβ.

The invention also relates to compositions which inhibit the interactionof RSK3 and mAKAPβ. In particular embodiments, these inhibitingcompositions or “inhibitors” include peptide inhibitors, which can beadministered by any known method, including by gene therapy delivery. Inother embodiments, the inhibitors can be small molecule inhibitors.

Specifically, the present invention is directed to methods andcompositions for treating or protecting the heart from damage, byadministering to a patient at risk of such damage, a pharmaceuticallyeffective amount of a composition which (1) inhibits the interaction ofRSK3 and mAKAPβ; (2) inhibits the activity of RSK3 and mAKAPβ; or (3)inhibits the expression of RSK3 and mAKAPβ.

The invention also relates to methods of treating or protecting theheart from damage, by administering to a patient at risk of such damage,a pharmaceutically effective amount of a composition which inhibits acellular process mediated by the anchoring of RSK3 through itsN-terminal domain.

In one embodiment, the composition includes an RSK3 peptide. In apreferred embodiment, the RSK3 peptide is obtained from the aminoterminus of the RSK3 amino acid sequence. In a particularly preferredembodiment, the RSK3 peptide is amino acids 1-42 of the RSK3 amino acidsequence.

In another embodiment, the composition includes a small interfering RNAsiRNA that inhibits the expression of either or both of RSK3 and mAKAPβ.In a preferred embodiment, the siRNA that inhibits the expression ofmAKAPβ is generated in vivo following administration of a short hairpinRNA expression vector or biologic agent (shRNA).

The composition of the invention can be administered directly or can beadministered using a viral vector. In a preferred embodiment, the vectoris adeno-associated virus (AAV).

In another embodiment, the composition includes a small moleculeinhibitor. In preferred embodiments, the small molecule is SL0101 orBI-D1870.

In another embodiment, the composition includes a molecule that inhibitsthe binding, expression or activity of mAKAPβ. In a preferredembodiment, the molecule is a mAKAPβ peptide. The molecule may beexpressed using a viral vector, including adeno-associated virus (AAV).

In yet another embodiment, the composition includes a molecule thatinterferes with RSK3-mediated cellular processes. In preferredembodiments, the molecule interferes with the anchoring of RSK3 throughits N-terminal domain.

The invention also relates to diagnostic assays for determining apropensity for heart disease, wherein the binding interaction of RSK3and mAKAPβ is measured, either directly, or by measuring a downstreameffect of the binding of RSK3 and mAKAPβ. The invention also provides atest kit for such an assay.

In still other embodiments, the inhibitors include any molecule thatinhibits the expression of RSK3 and mAKAPβ, including antisense RNA,ribozymes and small interfering RNA (siRNA), including shRNA.

The invention also includes an assay system for screening of potentialdrugs effective to inhibit the expression and/or binding of RSK3 andmAKAPβ. In one instance, the test drug could be administered to acellular sample with the RSK3 and mAKAPβ, or an extract containing theRSK3 and mAKAPβ, to determine its effect upon the binding activity ofthe RSK3 and mAKAPβ, by comparison with a control. The invention alsoprovides a test kit for such an assay.

In preparing the peptide compositions of the invention, all or part ofthe RSK3 (FIG. 2) or mAKAP (FIG. 3) amino acid sequence may be used. Inone embodiment, the amino-terminal region of the RSK3 protein is used asan inhibitor. Preferably, at least 10 amino acids of the RSK3 aminoterminus are used. More preferably, about 18 amino acids of the RSK3amino terminus are used. Most preferably, amino acids from about 1-42 ofthe RSK3 amino terminus are used.

In other embodiments, at least 10 amino acids of the mAKAP sequence areused. More preferably, at least 25 amino acids of the mAKAP sequence areused. Most preferably, peptide segments from amino acids 1694-1833 ofmAKAP are used.

It should be appreciated that various amino acid substitutions,deletions or insertions may also enhance the ability of the inhibitingpeptide to inhibit the interaction of RSK3 and mAKAPβ. A substitutionmutation of this sort can be made to change an amino acid in theresulting protein in a non-conservative manner (i.e., by changing anamino acid belonging to a grouping of amino acids having a particularsize or characteristic to an amino acid belonging to another grouping)or in a conservative manner (i.e., by changing an amino acid belongingto a grouping of amino acids having a particular size or characteristicto an amino acid belonging to the same grouping). Such a conservativechange generally leads to less change in the structure and function ofthe resulting protein. A non-conservative change is more likely to alterthe structure, activity or function of the resulting protein. Thepresent invention should be considered to include sequences containingconservative changes, which do not significantly alter the activity, orbinding characteristics of the resulting protein.

The following is one example of various groupings of amino acids:

Amino acids with nonpolar R groups: Alanine, Valine, Leucine,Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine.

Amino acids with uncharged polar R groups: Glycine, Serine, Threonine,Cysteine, Tyrosine, Asparagine, Glutamine.

Amino acids with charged polar R groups (negatively charged at pH 6.0):Aspartic acid, Glutamic acid.

Basic amino acids (positively charged at pH 6.0): Lysine, Arginine,Histidine (at pH 6.0).

Another grouping may be those amino acids with phenyl groups:Phenylalanine, Tryptophan, Tyrosine.

Another grouping may be according to molecular weight (i.e., size of Rgroups): Glycine (75), Alanine (89), Serine (105), Proline (115), Valine(117), Threonine (119), Cysteine (121), Leucine (131), Isoleucine (131),Asparagine (132), Aspartic acid (133), Glutamine (146), Lysine (146),Glutamic acid (147), Methionine (149), Histidine (at pH 6.0) (155),Phenylalanine (165), Arginine (174), Tyrosine (181), Tryptophan (204).

Particularly preferred substitutions are:

-   -   Lys for Arg and vice versa such that a positive charge may be        maintained;    -   Glu for Asp and vice versa such that a negative charge may be        maintained;    -   Ser for Thr such that a free —OH can be maintained; and    -   Gln for Asn such that a free NH₂ can be maintained.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced a potential site for disulfide bridges with another Cys. AHis may be introduced as a particularly “catalytic” site (i.e., His canact as an acid or base and is the most common amino acid in biochemicalcatalysis). Pro may be introduced because of its particularly planarstructure, which induces β-turns in the protein's structure. Two aminoacid sequences are “substantially homologous” when at least about 70% ofthe amino acid residues (preferably at least about 80%, and mostpreferably at least about 90 or 95%) are identical, or representconservative substitutions.

Likewise, nucleotide sequences utilized in accordance with the inventioncan also be subjected to substitution, deletion or insertion. Wherecodons encoding a particular amino acid are degenerate, any codon whichcodes for a particular amino acid may be used. In addition, where it isdesired to substitute one amino acid for another, one can modify thenucleotide sequence according to the known genetic code.

Nucleotides and oligonucleotides may also be modified. U.S. Pat. No.7,807,816, which is incorporated by reference in its entirety, andparticularly for its description of modified nucleotides andoligonucleotides, describes exemplary modifications.

Two nucleotide sequences are “substantially homologous” or“substantially identical” when at least about 70% of the nucleotides(preferably at least about 80%, and most preferably at least about 90 or95%) are identical.

Two nucleotide sequences are “substantially complementary” when at leastabout 70% of the nucleotides (preferably at least about 80%, and mostpreferably at least about 90 or 95%) are able to hydrogen bond to atarget sequence.

The term “standard hybridization conditions” refers to salt andtemperature conditions substantially equivalent to 5×SSC and 65 C forboth hybridization and wash. However, one skilled in the art willappreciate that such “standard hybridization conditions” are dependenton particular conditions including the concentration of sodium andmagnesium in the buffer, nucleotide sequence length and concentration,percent mismatch, percent formamide, and the like. Also important in thedetermination of “standard hybridization conditions” is whether the twosequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standardhybridization conditions are easily determined by one skilled in the artaccording to well known formulae, wherein hybridization is typically10-20 C below the predicted or determined T_(m) with washes of higherstringency, if desired.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to prevent, and preferably reduce by at least about 30percent, more preferably by at least 50 percent, most preferably by atleast 90 percent, a clinically significant change in a cardiac myocytefeature.

The preparation of therapeutic compositions which contain polypeptides,analogs or active fragments as active ingredients is well understood inthe art. Typically, such compositions are prepared as injectables,either as liquid solutions or suspensions, however, solid forms suitablefor solution in, or suspension in, liquid prior to injection can also beprepared. The preparation can also be emulsified. The active therapeuticingredient is often mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanol,or the like and combinations thereof. In addition, if desired, thecomposition can contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents which enhance theeffectiveness of the active ingredient.

A polypeptide, analog or active fragment, as well as a small moleculeinhibitor, can be formulated into the therapeutic composition asneutralized pharmaceutically acceptable salt forms. Pharmaceuticallyacceptable salts include the acid addition salts (formed with the freeamino groups of the polypeptide or antibody molecule) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed from the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

The therapeutic compositions of the invention are conventionallyadministered intravenously, as by injection of a unit dose, for example.The term “unit dose” when used in reference to a therapeutic compositionof the present invention refers to physically discrete units suitable asunitary dosage for humans, each unit containing a predetermined quantityof active material calculated to produce the desired therapeutic effectin association with the required diluent; i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's immune system to utilize the active ingredient, and degree ofinhibition of RSK3-mAKAPβ binding desired. Precise amounts of activeingredient required to be administered depend on the judgment of thepractitioner and are peculiar to each individual. However, suitabledosages may range from about 0.1 to 20, preferably about 0.5 to about10, and more preferably one to several, milligrams of active ingredientper kilogram body weight of individual per day and depend on the routeof administration. Suitable regimes for initial administration andbooster shots are also variable, but are typified by an initialadministration followed by repeated doses at one or more hour intervalsby a subsequent injection or other administration. Alternatively,continuous intravenous infusion sufficient to maintain concentrations often nanomolar to ten micromolar in the blood are contemplated.

Because of the necessity for the inhibitor to reach the cytosol, apeptide in accordance with the invention may need to be modified inorder to allow its transfer across cell membranes, or may need to beexpressed by a vector which encodes the peptide inhibitor. Likewise, anucleic acid inhibitor (including siRNAs, shRNAs and antisense RNAs) canbe expressed by a vector. Any vector capable of entering the cells to betargeted may be used in accordance with the invention. In particular,viral vectors are able to “infect” the cell and express the desired RNAor peptide. Any viral vector capable of “infecting” the cell may beused. A particularly preferred viral vector is adeno-associated virus(AAV).

siRNAs inhibit translation of target mRNAs via a process called RNAinterference. When the siRNA is perfectly complementary to the targetmRNA, siRNA act by promoting mRNA degradation. shRNAs, as a specializedtype of siRNA, have certain advantages over siRNAs that are produced asoligonucleotides. siRNA oligonucleotides are typically synthesized inthe laboratory and are delivered to the cell using delivery systems thatdeliver the siRNA to the cytoplasm. In contrast, shRNAs are expressed asminigenes delivered via vectors to the cell nucleus, where followingtranscription, the shRNA are processed by cellular enzymes such asDrosha and Dicer into mature siRNA species. siRNAs are usually 99%degraded after 48 hours, while shRNAs can be expressed up to 3 years.Moreover, shRNAs can be delivered in much lower copy number than siRNA(5 copies vs. low nM), and are much less likely to produce off-targeteffects, immune activation, inflammation and toxicity. While siRNAs aresuitable for acute disease conditions where high doses are tolerable,shRNAs are suitable for chronic, life threatening diseases or disorderswhere low doses are desired.(http://www.benitec.com/technology/sirna-vs-shrna)

Guidelines for the design of siRNAs and shRNAs can be found in Elbashir(2001) and at various websites includinghttps://www.thermofisher.com/us/en/home/references.ambion-tech-support/rnai-sirna/general-articles/-sirna-design-guidelines.htmland http://www.invivogen.com/review-sirna-shrna-design, all of which arehereby incorporated by reference in their entireties. Preferably, thefirst nucleotide is an A or a G. siRNAs of 25-29 nucleotides may be moreeffective than shorter ones, but shRNAs with duplex length 19-21 seem tobe as effective as longer ones. siRNAs and shRNAs are preferably 19-29nucleotides. Loop sequences in shRNAs may be 3-9 nucleotides in length,with 5, 7 or 9 nucleotides preferred.

With respect to small molecule inhibitors, any small molecule thatinhibits the interaction of RSK3 and mAKAPβ may be used. In addition,any small molecules that inhibit the activity of RSK3 and/or mAKAPβ maybe used. Particularly preferred small molecules include BI-D1870,available from Enzo Life Sciences

and SL0101, available from Millipore:

Small molecules with similar structures and functionalities can likewisebe determined by rational and screening approaches.

Likewise, any small molecules that inhibit the expression of RSK3 and/ormAKAPβ may be used.

In yet more detail, the present invention is described by the followingitems which represent preferred embodiments thereof:

-   -   1. A composition comprising a vector encoding a small hairpin        ribonucleic acid (“shRNA”) against human mAKAP.    -   2. The composition of item 1, wherein the shRNA inhibits the        interaction between human RSK3 and human mAKAP.    -   3. The composition of item 1, wherein a therapeutically        effective amount of said composition administered to a patient        in need thereof protects the patient from heart damage.    -   4. The composition of item 1, wherein the vector is a viral        vector.    -   5. The composition of item 4, wherein the viral vector is an        adeno-associated virus vector (AAV).    -   6. The composition of item 1, wherein the shRNA comprises a        sense copy of a human mAKAP mRNA sequence and an antisense copy        of a human mAKAP mRNA sequence.    -   7. The composition of item 6, wherein the sense and antisense        copies of the human mAKAP mRNA sequence are separated by a loop        sequence.    -   8. The composition of item 5, wherein the sense and antisense        copies of the human mAKAP mRNA sequence are 19 nucleotides in        length.    -   9. The composition of item 1, wherein the vector comprises a        sequence substantially complementary to one of the following        nucleotide sequences:

(SEQ ID NO: 7) GGTTGAAGCTTTGAAGAAA, (SEQ ID NO: 8) GCTAAGAGATACAGAGCTTor (SEQ ID NO: 9) GGAGGAAATAGCAAGGTTA.

-   -   10. The composition of item 9, wherein the vector comprises one        of the following nucleotide sequences:

(SEQ ID NO: 7) GGTTGAAGCTTTGAAGAAA, (SEQ ID NO: 8) GCTAAGAGATACAGAGCTTor (SEQ ID NO: 9) GGAGGAAATAGCAAGGTTA.

-   -   11. The composition of item 10, wherein the vector comprises the        following nucleotide sequence:

(SEQ ID NO: 27) GGAGGAAATAGCAAGGTTA.

-   -   12. The composition of item 4, wherein the viral vector        comprises human microRNA (miR)-30a sequences.    -   13. The composition of item 4, wherein the viral vector        comprises a shRNA minigene, wherein the minigene is flanked by a        complete AAV2 inverted terminal repeat (ITR) on the 5′ end and a        deleted AAV2 ITR on the 3′ end.    -   14. The composition of item 4, wherein the viral vector        comprises a chicken cardiac troponin T promoter.    -   15. The composition of item 4, wherein the viral vector        comprises a SV40 polyadenylation site.    -   16. The composition of item 5, wherein the AAV is an AAV        serotype 9.    -   17. A method of inhibiting the expression of human mAKAPβ,        comprising contacting mRNA encoding mAKAPI3 with the composition        of item 1.    -   18. A method of protecting the heart from damage, comprising        administering to a patient at risk of such damage, a        pharmaceutically effective amount of the composition of item 1.    -   19. A method of treating or preventing heart disease, comprising        administering to a patient in need thereof, a pharmaceutically        effective amount of the composition of item 1.    -   20. A method of treating or preventing myocardial infarction,        comprising administering to a patient at risk of such damage, a        pharmaceutically effective amount of the composition of item 1.

The following examples are provided to aid the understanding of thepresent invention, the true scope of which is set forth in the appendedclaims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

EXAMPLES

The compositions and processes of the present invention will be betterunderstood in connection with the following examples, which are intendedas an illustration only and not limiting of the scope of the invention.Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art and such changes and modificationsincluding, without limitation, those relating to the processes,formulations and/or methods of the invention may be made withoutdeparting from the spirit of the invention and the scope of the appendedclaims.

Example 1

Methods

Reagents

Commercial antibodies and oligonucleotides are listed in FIG. 19 andFIG. 20 of U.S. Pat. No. 9,132,174. The commercially available RSK3antibodies were of varying specificity (FIG. 21 of U.S. Pat. No.9,132,174). Additional reagents and detailed methods are provided.

RSK3^(−/−) Mouse

All experiments involving animals were approved by the InstitutionalAnimal Care and Use Committee at the University of Miami. ConstitutiveRSK3 knockout mice were backcrossed to C57BL/6 mice over 10 generations.All experiments were performed with littermate controls and mice thatwere 8 to 10 weeks of age at the beginning of the study. Transverseaortic constriction was performed as previously described (Rockman1991), and isoproterenol infusion was via Alzet 2002 osmotic pumps(Durect). Echocardiography was performed under isoflurane anesthesia ona Vevo 770 High-Resolution Imaging System (VisualSonics).

RNA Assays

mRNA species were assayed using NanoString technology, a direct andmultiplexed measurement of gene expression without amplification, usingfluorescent molecular bar codes and single-molecule imaging to identifyand count multiple transcripts in a single reaction; 100 ng of RNA washybridized in solution to a target-specific code set overnight at 65°C., and individual mRNAs were counted using a NanoString DigitalAnalyzer.

Statistics

For all experiments, n refers to the number of individual mice orindividual myocyte preparations. All data are expressed as mean±SEM. Pvalues were calculated using Student t tests and are not corrected formultiple comparisons. Repeated symbols represent P values of differentorders of magnitude (i.e., P<0.05, P<0.005, and others). All datasetsinvolving multiple comparisons for which P values are provided also weresignificant by ANOVA (α=0.05).

Results

mAKAPβ: A Scaffold for RSK

The inventors have previously published that RSK proteins and activityare associated with mAKAPα complexes in the brain (Michel 2005). Theinventors now show that RSK also is associated with mAKAPβ in cardiacmyocytes (FIG. 4B of U.S. Pat. No. 9,132,174). To determine whethermAKAP preferentially binds a specific RSK isoform, hemagglutinin(HA)-tagged RSK family members were co-expressed with mAKAP in HEK293cells. In contrast to RSK1 and RSK2, RSK3 robustly mediated thecoimmunoprecipitation of both mAKAPα and mAKAPβ (FIG. 4C of U.S. Pat.No. 9,132,174). RSK family members are similar in primary sequence withthe exception of the extreme N-terminal and C-terminal domains and asmall region after the hydrophobic motif (FIG. 4A of U.S. Pat. No.9,132,174). Consistent with the selective binding of RSK3 to thescaffold, the N-terminal domain of RSK3 bound mAKAPβ (FIG. 4D of U.S.Pat. No. 9,132,174).

RSK3 Function in Neonatal Cardiac Myocytes

RSK family members can be activated in most cell types by ERK, but notc-Jun N-terminal kinases or p38 (Anjum 2008). ERK phosphorylation ispermissive for PDK1 phosphorylation of the RSK N-terminal kinase domain,such that PDK1 phosphorylation of RSK S²¹⁸ is indicative of fullactivation of the enzyme (FIG. 4A of U.S. Pat. No. 9,132,174). To showthat ERK activates RSK in cardiac myocytes, the inventors treatedneonatal rat ventricular myocytes with different hypertrophic agonistsand mitogen-activated protein kinase pathway inhibitors and detected RSKactivation using a pan-RSK S²¹⁸ phosphor-specific antibody (FIGS. 21 and22 of U.S. Pat. No. 9,132,174). The α-adrenergic stimulation withphenylephrine (PE) induced RSK phosphorylation 3-fold by bothMEK1/2-dependent (that activates ERK1/2) and MEK5-dependent (thatactivates ERK5) mechanisms (FIG. 22 of U.S. Pat. No. 9,132,174).Moreover, MEK1/2 inhibition reduced RSK baseline phosphorylation. c-JunN-terminal kinase and p38 inhibition did not affect PE activation and,in fact, variably increased baseline RSK phosphorylation. Fetal bovineserum and leukemia inhibitory factor also increased the level ofactivated RSK, but that occurred more so because of an increase in totalRSK protein expression than because of ERK phosphorylation.

Similar results were found for HA-tagged RSK3 (FIG. 5A of U.S. Pat. No.9,132,174). Acute PE treatment induced the phosphorylation of HA-RSK3ERK (S³⁶⁰) and PDK1 (S²¹⁸) sites through both MEK1/2-dependent andMEK5-dependent signaling. Together, these results confirmed that incardiac myocytes ERK is responsible for RSK activation.

The inventors have previously demonstrated that mAKAPβ complexes arerequired for the hypertrophy of cultured myocytes (Li 2010, Pare 2005,Dodge-Kafka 2005). Therefore, the inventors proposed the hypothesis thatRSK3 signaling is a major determinant of cardiac myocyte growth.Neonatal myocytes were transfected with small interfering RNAoligonucleotides (siRNA) that diminished RSK3 mRNA and protein levelsby >75% (FIG. 5B of U.S. Pat. No. 9,132,174). RSK3 siRNA did not inducethe apoptosis of myocytes cultured either in the absence or in thepresence of serum (FIG. 5C of U.S. Pat. No. 9,132,174). Importantly, inthe presence of α-adrenergic stimulation, RSK3 siRNA inhibitedmorphologic hypertrophy by 34% and atrial natriuretic factor expressioncompletely (FIG. 5D-5F and FIG. 23 of U.S. Pat. No. 9,132,174). Inaddition, RSK3 siRNA had smaller, but detectable, effects on leukemiainhibitory factor and fetal bovine serum-stimulated hypertrophy. Theresults obtained by RSK3 RNA interference were confirmed with a seconddistinct RSK3 siRNA.

Endogenous RSK3 proteins are expressed at a relatively low level incardiac myocytes compared with the other RSK family members and areinduced in expression by long-term PE treatment (FIG. 24C of U.S. Pat.No. 9,132,174). As a result, RSK3 RNA interference did not affect thelevel of total RSK in the myocyte, only diminishing the RSK3 detectedafter immunoprecipitation with a specific RSK3 antibody (FIG. 5B andFIG. 24C of U.S. Pat. No. 9,132,174). In control experiments, theinhibition of PE-induced hypertrophy by the RSK3 siRNA was rescued bythe expression of recombinant HA-tagged human RSK3, but not by aninactive HA-RSK3 S²¹⁸A mutant (FIGS. 24A and 24B of U.S. Pat. No.9,132,174). Remarkably, in these experiments, the cross-section area ofunstimulated myocytes was increased by adenoviral-based expression ofwild-type HA-RSK3 enzyme at a level comparable with that of endogenousRSK3 in PE-treated cells without affecting total RSK levels. Finally, toconfirm that RSK activity was important for neonatal myocytehypertrophy, the inventors used the pan-RSK inhibitors BI-D1870 (FIG. 6and FIG. 25 of U.S. Pat. No. 9,132,174) and SL0101 (FIG. 26 of U.S. Pat.No. 9,132,174) (Smith 2005, Sapkota 2007, Malone 2005), finding that,like RSK3 siRNA, these compounds inhibited agonist-induced myocytehypertrophy.

High-Affinity RSK3 Binding Domain in mAKAP

The inventors considered that the requirement for RSK3 in myocytehypertrophy was attributable to the association of RSK3 with specificsignaling complexes. To address this hypothesis, the inventors definedthe mAKAP domains responsible for RSK3 binding. HA-tagged RSK3 wasco-expressed in heterologous cells with myc-tagged mAKAPβ fragments andcoimmunoprecipitated using a myc-tag antibody (FIGS. 7A and 7B of U.S.Pat. No. 9,132,174). RSK3 preferentially associated with mAKAP aminoacid residues 1286 to 1833, although it also weakly associated withmAKAP 245 to 587 and 525 to 1286. Consistent with this result, RSK3binding to a full-length mAKAPβ protein with an internal deletion ofresidues 1257 to 1886 was reduced by >85%. Further mapping showed thatthe main RSK3 binding domain (RBD) of mAKAP mapped to a fragmentencompassing residues 1694 to 1833 (FIG. 7C of U.S. Pat. No. 9,132,174).Accordingly, RSK3 bound poorly to a full-length mAKAPβ protein with aninternal deletion of residues 1701 to 1800 (FIG. 7D of U.S. Pat. No.9,132,174). As shown, the unique N-terminal domain of RSK3 boundfull-length mAKAPβ (FIG. 4D of U.S. Pat. No. 9,132,174). The mAKAP RBDalso bound HA-RSK3 1 to 42 (FIG. 7E of U.S. Pat. No. 9,132,174), but notto the N-terminally truncated RSK3 mutant (HA-RSK3 DN30) or theHA-tagged full-length RSK2 (FIG. 7F of U.S. Pat. No. 9,132,174). Theseresults imply that the mAKAP RBD is responsible for the selectivebinding of RSK3 to mAKAP.

The inventors next tested whether mAKAP-RSK3 binding is direct (FIG. 7Gof U.S. Pat. No. 9,132,174). The binding of bacterially expressedHis-tagged mAKAP 1286 to 1833 and full-length RSK3 was analyzed bysurface plasmon resonance. The binding was direct and of high affinity(nanomolar K_(D)). The inventors previously reported that onceactivated, RSK3 binds mAKAPα less well in cells (Michele 2005).Interestingly, previous RSK3 phosphorylation by either ERK or both ERKand PDK1 decreased the RSK3 binding affinity for mAKAP 5-fold and8-fold, respectively, through a decrease in the association rateconstant.

Disruption of RSK3 Anchoring Inhibits Neonatal Myocyte Hypertrophy Theidentification of the high-affinity mAKAP RBD provided the opportunityto test whether anchoring of RSK3 is important for its function. Whenexpressed in neonatal myocytes, a green fluorescent protein—mAKAP RBDfusion protein competed the association of endogenous RSK3 and mAKAPβ(FIG. 8A of U.S. Pat. No. 9,132,174). Expression of green fluorescentprotein—mAKAP RBD fusion protein markedly inhibited PE-inducedhypertrophy (FIG. 8B-5D of U.S. Pat. No. 9,132,174), similar to RSK3siRNA (FIG. 5 of U.S. Pat. No. 9,132,174). Together, these results implythat RSK3 anchored to scaffolds through its unique N-terminal domain isrequired for the hypertrophy of cultured myocytes.

Role of RSK3 in Cardiac Hypertrophy In Vivo

The results obtained in vitro suggested that active RSK3 contributes tothe development of pathologic myocyte hypertrophy. Further evidencesupporting this hypothesis was obtained using a new RSK3 knockout mouse.By homologous recombination, stop codons were inserted into the secondexon of the RSK3 (Rps6ka2) gene encoding the ATP-binding motif of theN-terminal kinase domain (Hanks 1988), resulting in the constitutiveabsence of RSK3 protein in homozygous null mice (FIG. 27 of U.S. Pat.No. 9,132,174). In general, RSK3^(−/−) mice appeared normal inmorphology, were bred according to Mendelian genetics (FIG. 12 of U.S.Pat. No. 9,132,174), and exhibited no excess mortality up to 6 months ofage. Before any stress, the RSK3^(−/−) mice had generally normal cardiacfunction, with the only measureable difference from wild-typelittermates being a slight increase in left ventricular internaldimensions detected by echocardiography (FIGS. 13 and 28 of U.S. Pat.No. 9,132,174).

The inventors tested whether RSK3 is required for compensated cardiachypertrophy by subjecting the RSK3^(−/−) mice to pressure overload for 2weeks (FIG. 9A of U.S. Pat. No. 9,132,174). By echocardiography,transverse aortic constriction (TAC) induced a 36% increase in posteriorwall thickness in wild-type mice, but only a 16% increase in RSK3^(−/−)mice (FIGS. 10 and 28 of U.S. Pat. No. 9,132,174). The decreasedhypertrophy was not accompanied by a change in contractility (fractionalshortening). Postmortem gravimetric analysis showed that thecorresponding increase in biventricular weight after TAC was similarlydiminished in the knockout mice (48% for RSK3⁺⁺ vs. 26% for RSK3^(−/−)mice; FIG. 14 of U.S. Pat. No. 9,132,174). TAC primarily inducesconcentric growth of cardiac myocytes.

Inspection of wheat germ agglutinin-stained heart sections revealed thatconsistent with these results, RSK3 knockout attenuated the TAC-inducedincrease in myocyte transverse cross-section area by ˜46% (FIGS. 9B and9C of U.S. Pat. No. 9,132,174). Proportional results were obtained bymorphometric analysis of adult cardiac myocytes isolated from the TACmice (FIG. 9D-9G of U.S. Pat. No. 9,132,174).

To characterize the RSK3^(−/−) cardiac phenotype at a molecular level,the inventors surveyed for differences in the cardiac expression of 30genes encoding proteins involved either in cardiac remodeling or inhypertrophic signaling (FIG. 11 of U.S. Pat. No. 9,132,174).Approximately two-thirds of the genes in our panel were significantlyincreased or decreased in expression by TAC. In general, the changes inexpression were attenuated by RSK3 knockout. For example, TAC-inducedatrial natriuretic factor expression was dramatically inhibited inRSK3^(−/−) mice, consistent with the results obtained for PE-treatedneonatal myocytes. Although after 2 weeks of pressure overload the smallincreases in cellular apoptosis and interstitial fibrosis detectable byhistology for wild-type mice did not reach significance when comparedwith sham-operated controls, these signs of remodeling tended to be lessin the knockout mice (8.2±2.0 vs. 4.2±1.0×10⁻⁴ TUNEL-positive nuclei and0.49%±0.18% vs. 0.29%±0.11% collagen staining for wild-type andRSK3^(−/−) TAC hearts, respectively). Interestingly, 2 genetic markersof fibrosis that were significantly induced in TAC wild-type mice,transforming growth factor β2 and collagen VI α1 (Yang 2012), wereattenuated in expression by RSK3 knockout (FIG. 11 of U.S. Pat. No.9,132,174).

To further explore the role of RSK3 in cardiac hypertrophy, theinventors used a second in vivo pathological stressor, chronicisoproterenol (Iso) infusion via subcutaneous osmotic pumps, and aphysiological stressor, chronic exercise via swimming. Although Isoinfusion resulted in a minor increase in ventricular wall thickness byechocardiography (FIG. 15 of U.S. Pat. No. 9,132,174), at the cellularlevel Iso significantly induced myocyte growth in width in aRSK3-dependent manner as measured by histology and after myocyteisolation (FIG. 29 of U.S. Pat. No. 9,132,174). Unlike TAC, Iso infusionalso induced eccentric growth, as evidenced by increased myocyte lengthand ventricular dilation by echocardiography (FIGS. 15 and 16 of U.S.Pat. No. 9,132,174). This eccentric growth was not inhibited by RSK3knockout. Together with the TAC data, these results demonstrate thatRSK3 contributes to the induction of concentric myocyte hypertrophy inpathologic conditions.

Finally, RSK3^(−/−) mice were exercised by swimming. As expected(Perrino 2006), after swimming, wild-type mice exhibited a decreasedresting heart rate (consistent with improved physical conditioning) andincreased left ventricular internal dimensions (FIGS. 17 and 28 of U.S.Pat. No. 9,132,174). After exercise, there were no significantdifferences between RSK3 knockout and wild-type mice detectable byechocardiography, and the cohorts exhibited a similar increase inbiventricular weight indexed by body weight (6% and 7%, respectively;FIG. 18 of U.S. Pat. No. 9,132,174).

Detailed Methods

Reagents: Commercial antibodies are listed in FIG. 19 of U.S. Pat. No.9,132,174. Secondary antibodies included horseradish peroxidase(HRP)-conjugated donkey secondary antibodies (Jackson ImmunoResearch)and Alexa dye-conjugated donkey secondary antibodies (Invitrogen).Monoclonal 211, polyclonal VO54, VO56 and OR010 mAKAP antibodies were aspreviously described and are available through Covance Research Products(Kehat 2010). OR42 and OR43 rabbit anti-RSK3 antisera were generatedusing bacterially-expressed His-tagged RSK3 (full-length) and affinitypurified using antigen-coupled Affigel resin (Biorad). FL099 and FL100rabbit anti-mAKAP antisera were generated using bacterially-expressedGST-tagged mAKAP 245-340. Oligonucleotides are listed in FIG. 20 of U.S.Pat. No. 9,132,174. Other reagents included: BIX02189-BoehringerIngelheim Pharmaceuticals; PD0325901, SB103580, SL0101, and SP600125-EMDChemicals Inc.

All adenovirus were constructed using the pTRE shuttle vector and theAdeno-X Tet-off System (Clontech) and purified after amplification usingVivapure AdenoPACK kits (Sartorius Stedim). These adenovirusesconditionally express recombinant protein when co-infected withtetracycline transactivator-expressing virus (adeno-tTA for “tet-off” orreverse tTA for “tet-on”). HA-tagged RSK and MSK2 expression plasmidsacquired from Dario Alessi and John Blenis (McKinsey 2007, Anjum 2008,Sadoshima 1995) and myc-tagged mAKAP mammalian expression vectors(pCDNA3.1 (−) myc-his) and adenovirus were as previously described(Kodama 2000, Takeishi 2002). GFP-RBD was expressed using a pEGFP-basedplasmid. Bacterial expression vectors for mAKAP and RSK3 wereconstructed using pET30 and pGEX-4T parent vectors, and proteins werepurified using His-bind (Novagen) and Glutathione Uniflow Resins(Clontech).

Neonatal rat myocytes isolation and culture: 1-3 day old Sprague-Dawleyrats were decapitated and the excised hearts placed in 1×ADS Buffer (116mmol/L NaCl, 20 mmol/L HEPES, 1 mmol/L NaH₂PO₄, 5.5 mmol/L glucose, 5.4mmol/L KCl, 0.8 mmol/L MgSO₄, pH 7.35). The atria were carefully removedand the blood washed away. The ventricles were minced and incubated with15 mL 1×ADS Buffer containing 3.3 mg type II collagenase (Worthington,230 U/mg) and 9 mg Pancreatin (Sigma) at 37° C. while shaking at 80 RPM.After 15 minutes, the dissociated cardiac myocytes were separated bycentrifugation at 50×g for 1 minute, resuspended in 4 mL horse serum andincubated 37° C. with occasional agitation. The steps for enzymaticdigestion and isolation of myocytes were repeated 10-12 times tomaximize yield. The myocytes were pooled and spun down again at 50×g for2 minutes and resuspended in Maintenance Medium (DMEM:M199, 4:1)supplemented with 10% horse serum and 5% fetal bovine serum. To removeany contaminating fibroblasts, the cells were pre-plated for 1 hourbefore plating on gelatin-coated tissue culture plasticware. Thisprocedure yields >90% pure cardiac myocytes. After 1 day in culture, themedia was changed to maintenance medium containing 0.1 mmol/Lbromodeoxyuridine to suppress fibroblast growth.

Experiments were initiated 1 day after myocyte isolation. Adenoviralinfection was performed by addition of adenovirus (multiplicity ofinfection=5-50) to the media. Plasmids and siRNA oligonucleotides weretransfected using Transfast (Promega) and Dharmafect (Thermofisher),respectively, as recommended by the manufacturers using cells culturedin maintenance medium supplemented with 4% horse serum. Starting the dayafter gene transduction, the cells were treated for as long as 2 days,as indicated for each experiment.

Immunoprecipitations: HEK293 and COS-7 cells were transfected withLipofectamine 2000 (Invitrogen) or Polyethylenimine “Max”(Polysciences). Cells (including myocytes) were lysed in buffer (20mmol/L HEPES, pH 7.4, 150 mmol/L NaCl, 5 mmol/L EDTA, 0.5% Triton, 50mmol/L NaF, 1 mmol/L sodium orthovanadate, 1 mmol/L DTT, and proteaseinhibitors). After centrifugation at 10,000×g for 10 minutes at 4° C.,the clarified extracts were used for immunoprecipitation usingappropriate antibodies (10 μg purified antibody or 1-5 μL whole serum)and 20 μL protein G sepharose (Millipore, Fastflow) for 3 hours toovernight at 4° C. The beads were washed 3-5 times with lysis buffer,and the immunoprecipitated proteins were eluted with 1× Laemmli bufferfor western blotting. Western blots were developed using horseradishperoxidase-conjugated donkey secondary antibodies, Supersignal WestChemiluminescent Substrates (Thermo Scientific) and X-ray film or aFujifilm LAS-3000 imaging system.

Immunocytochemistry: Cultured neonatal cardiomyocytes on plasticcoverslips were fixed in 3.7% formaldehyde in PBS, permeabilized with0.3% Triton X-100 in PBS, and blocked with PBS containing 0.2% BSA and1% horse serum for 1 hour. The slides were then sequentially incubatedfor 1 hour with primary and Alexa fluorescent dye-conjugatedspecific-secondary antibodies (Invitrogen, 1:1000) diluted in blockingbuffer. The slips were washed three times with blocking buffer. 1 μg/mLHoechst 33258 was included in the last wash stop to label nuclei. Slideswere sealed in SlowFade Gold antifade buffer (Invitrogen) forfluorescent microscopy. Wide-field images were acquired using a LeicaDMI 6000 Microscope.

Surface Plasmon Resonance: SPR analysis was performed using a BIAcoreT100. 200 resonance units His-tagged mAKAP 1286-1833 were covalentlyimmobilized using NHS (N-hydroxysuccinamide) and EDC[1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide] (Biacore aminecoupling kit) to the surface of a sensor chip (BIAcore type CM5).His-RSK3 analytes (6.25-200 nmol/L) in HBS buffer (10 mmol/L Hepes, pH7.4, 150 mmol/L NaCl, and 0.005% Surfactant P20) were injected at a flowrate of 30 μL/min for 5 minutes, followed by buffer alone for another 5minutes. Sensorgrams were processed by BIAcore T100 evaluation software.

For phosphorylated His-RSK3, 20 μg His-RSK3 was phosphorylated with 2 μgERK2 (Millipore, 14-550) and/or PDK1 (Sigma, P7498) for 5 hours inkinase buffer (20 mmol/L MOPS, pH 7.2, 25 mmol/L β-glycerol phosphate, 5mmol/L EGTA, 1 mmol/L sodium orthovanadate, 1 mmol/L DTT) with 0.5mmol/L MgATP. Phospho-His-RSK3 was purified using His-binding beads andconcentrated before use.

Generation of RSK3^(−/−) mouse: All experiments involving animals wereapproved by the Institutional Animal Care and Use Committee at theUniversity of Miami. Constitutive knock-out mice were generated using atargeting vector that inserted into Exon 2 a neomycin resistance gene(PGKneo) flanked by loxp sites (FIG. 27 of U.S. Pat. No. 9,132,174).Targeted 129SvJ ES cells were injected into C57BL/6J blastocysts. PGKneowas removed by crossing mutant mice with B6.C-Tg(CMV-cre)1Cgn/J (TheJackson Laboratory). RSK^(+/−) Mice were selected for loss of the cretransgene and backcrossed to C57BL/6 mice over 10 generations. Allexperiments were performed with littermate controls and mice that were8-12 weeks of age. The numbers of mice in each cohort are listed in thevarious tables and figures.

Isoproterenol infusion: Alzet 2002 osmotic pumps (Durect) were sterilelyloaded with 200 μL saline or saline and isoproterenol to deliver 60mg/kg/day for 14 days. 8 week old mice were anaesthetized, and the pumpwas inserted sterilely subcutaneously into the shaved back through atransverse incision made intra-scapulae. The wound was closed withsurgical staples and covered with betadine solution. Mice were housedseparately after surgery.

Transverse Aortic Constriction: All tools were sterilized with aGerminator 500 Dry Sterilizer and Betadine Solution (10% povidone-iodinetopical solution). Anesthesia was induced with 5% isoflurane andmaintained with 2% isoflurane and 100% oxygen at a flow rate of 1.5L/min using a SurgiVet flow regulator via nose cone. Loss ofconsciousness was verified by toe pinch. Mouse fur over the left chestand sternum was removed with a calcium hydroxide lotion (e.g. Nair), andthe surgical site was sterilized with betadine. The skin was incisedexposing the pectoralis muscle and the second left intercostal space.The pectoralis muscle and the second rib were blunt dissected andretracted revealing the thymus within the mediastinum. The lobes of thethymus were retracted to reveal the transverse aortic arch as well asthe right innominate and left common carotid arteries. Blunt dissectionthough the connective tissue between these two arteries and under theaorta allowed for the passage of a 6-0 silk using a modified ligationaid (Fine Science Tools 18062-12). A 27 gauge needle was placed on topof the aorta and the 6-0 silk was tied around the needle. The needle wasremoved, leaving a constricted aorta. The chest was closed in two layerswith 5-0 Polysorb Suture. Isoflurane administration was terminated, andthe mice were maintained on 100% oxygen by nose cone until conscious.Immediately post-operatively, buprenorphrine (0.05-0.1 mg/kg s.c.) wasadministered and then q12 h prn. The mice were allowed to recover undera heat lamp until alert and active. Sham-operated mice that experienceall but the placement of the aortic ligature served as controls.

Swimming: 8-10 week old mice were forced to swim in water tanks everyday for 4 weeks. The swimming tank measured >225 cm², with a depth of 15cm and a water temperature of 30-32° C. Mice were continuously observedto avoid any drowning. The first day of training consisted of two 10-minsessions separated by at least 4 h. Sessions were increased by 10 mineach day until 90-min sessions were reached. Additional cohorts werehoused normally without exercise to serve as a “sham swim” controlgroup. Food and water were provided ad libitum throughout the monthperiod for all mice.

Echocardiography: Mice minimally anesthetized with 1-2% isoflurane werestudied using a Vevo 770®, High-Resolution Imaging System(VisualSonics). The pressure gradient following TAC was calculated fromthe pulse wave Doppler velocity at the point of ligation as follows:P=4v²; P=the induced pressure gradient (in mmHg) and v=the velocityacross the constriction (in m/s).⁷

Adult mouse myocytes isolation by Langendorff perfusion: Mice wereanesthetized using Ketamine (80-100 mg/kg) and Xylazine (5-10 mg/kg) IPfollowed by 200 U heparin IP and cardiac excision. The heart was placedimmediately in perfusion buffer (NaCl 120 mmol/L, KCl 5.4 mmol/L,Na₂HPO₄.7H₂O 1.2 mmol/L, NaHCO₃ 20.0 mmol/L, MgCl₂.6H₂O 1.6 mmol/L,Taurine 5 mmol/L, Glucose 5.6 mmol/L) equilibrated with 95% O₂ and 5%CO₂. The heart was attached via the aorta to the condenser outlet of aHarvard Langendorff apparatus. Ca²⁺-free perfusion lasted for 5 minuteswith a constant rate at 2.2 mL/min at 37° C. The heart was digested bycontinuous perfusion with 25 mL buffer containing 25 mg type IIcollagenase (Worthington, 315 U/mg) and 1.3 mg protease (Sigma typeXIV). After removal of the atria, the ventricles were then immersed in 5mL of the same enzyme solution for dissociation by cutting into smallpieces and by passing through a large bore pipette. The cell slurry wasfiltered through a 150-200 μm nylon mesh and the myocytes relaxed byincubation for 10 minutes in perfusion buffer containing 10 mmol/L KCl.The cells were fixed in suspension in perfusion buffer containing 3.7%formaldehyde, before morphometric analysis by light microscopy.Histochemistry: Heart tissue was fixed in 3.7% formaldehyde.De-paraffinized 5 μm tissue sections were stained using the PicrosiriusRed Stain Kit (Polysciences) and Alexa Fluor 555 Wheat Germ Agglutininconjugate (Invitrogen) as recommended by the manufacturers. Thecross-section area of >150 myocytes in >3 distinct regions of the leftventricle were measured per heart using the wheat germ agglutininsections. Collagen content was assayed using the Picrosirius Red stainedsections and polarized light microscopy for >3 5× objective fields perheart. TUNEL staining for both fixed cells and tissue sections wasperformed using the In Situ Cell Death Detection Kit, TMR red (Roche).Morphometrics and collagen content were measured using IPLab microscopesoftware (BD Biosciences).

Morphometry: Morphometric data was acquired using IPLab Software. Forneonatal myocytes, at least 6 separate images, each containing >100cells, were assayed for cross-section area and perinuclear prepro-ANFstaining per condition for each repetition of the experiment. For adultmouse cardiac myocytes, the maximum lengths perpendicular (width) orparallel (length) to the myofibrils were measured for >100 freshlydissociated myocytes per heart.

RNA Assays: Total RNA was quantified with a Nanodrop 8000Spectrophotometer (Thermo Scientific) and quality controlled using witha Bioanalyzer 2100 and the RNA 6000 Nano kit (Agilent). qRT-PCR wasperformed using SYBR green.

The NanoString assay is based on direct, multiplexed measurement of geneexpression without amplification, utilizing fluorescent molecularbarcodes and single molecule imaging to identify and count multipletranscripts in a single reaction. For Nanostring assay, 100 ng total RNAwere hybridized in solution to a target-specific codeset overnight at65° C. The codeset contained dual, adjacently placed 50 bpoligonucleotide probes against a panel of 30 genes, one set of probesfluorescently bar-coded and the other biotinylated. The hybridizationreactions were loaded onto the NanoString Prep station which removesexcess oligonucleotides and binds the hybridized mRNA to theStreptavidin-coated cartridge surface. The cartridges were loaded ontothe NanoString Digital Analyzer, and 1155 fields of view werefluorescently scanned to count only those individual mRNAs bound to botha biotinylated and fluorescently bar-coded probe. Datasets for each RNAsample were background-subtracted and normalized using Gapdh. Invalidation assays, NanoString counts were directly proportional over 3orders of magnitude to the mRNA levels obtained by qRT-PCR and had asimilar minimum level of detection.

Statistics: For all experiments, n refers to the number of individualmice or individual myocyte preparations. All data are expressed asmean±s.e.m. p-values were calculated using two-tailed Student's t-tests,paired or un-paired as appropriate, and are not corrected for multiplecomparisons. Repeated symbols represent p-values of different orders ofmagnitude, for example: * p<0.05, ** p<0.005, *** p<0.0005, etc. Alldatasets involving multiple comparisons for which p-values are providedwere also significant by ANOVA, α0.05.

Discussion

RSK activity is associated with the function of the nervous system,immunity, muscle, and cancer (Anjum 2008). Human RSK2 mutations causeX-linked Coffin-Lowry syndrome, which includes mental and growthretardation and skeletal and facial anomalies, but rare cardiacabnormality. In the heart, RSK1 and RSK2 can activate the Na⁺/H⁺exchanger NHE1, and α-adrenergic-induced NHE1 phosphorylation is blockedby fmk, which inhibits all RSKs except RSK3 (Cuello 2007). The inventorsnow reveal a role for RSK3 in the cardiovascular system, regulation ofpathological myocyte hypertrophy.

Cardiac myocytes can grow in both width and length, termed concentricand eccentric hypertrophy, respectively (Kehat 2010). Concentric myocytehypertrophy involves the parallel assembly of contractile units(sarcomeres), increasing potential myocyte tension and wall thickness.In contrast, eccentric myocyte hypertrophy involves the serial assemblyof sarcomeres along the axis of contraction, mainly contributing toincreased ventricular wall area. The inventors found that RSK3 wasrequired for TAC-induced concentric hypertrophy, as well as forIso-induced myocyte growth in width in vivo. These differences can bemodeled in vitro. Interleukin-6 cytokines such as leukemia inhibitoryfactor and cardiotrophin-1 induce an elongated and eccentric phenotypefor cultured neonatal myocytes, in contrast to the symmetric growthstimulated by PE (Wollert 1996). Interestingly, the growth of thecultured myocytes tended to depend on RSK3 more when induced byα-adrenergic stimulation than by leukemia inhibitory factor (FIGS. 5 and6 of U.S. Pat. No. 9,132,174). The greater inhibition of PE-inducedmorphologic hypertrophy was consistent with the more robust activationof RSK by PE than leukemia inhibitory factor (FIG. 22 of U.S. Pat. No.9,132,174), as well as the results obtained in vivo.

RSK3 was activated in myocytes by ERK1, ERK2, and ERK5 (FIG. 5A of U.S.Pat. No. 9,132,174). Whereas RSK3 has been absent from the cardiacliterature, ERK signaling has been well-studied both in human diseaseand in animal models. The autosomal-dominant human syndromes Noonan,Costello, cardiofaciocutaneous, and LEOPARD result from mutations inPTPN11, HRAS, RAF1, BRAF, MEK1, and MEK2 that activate ERK1/2 signaling(Wu 2011). These Rasopathies feature developmental delay, dysmorphicfeatures, and defects in multiple organ systems, often including ahypertrophic phenotype Gelb 2011). In mice, left ventricular hypertrophyhas been induced by cardiac myocyte-specific expression ofconstitutively active H-Ras and MEK1, as well as cardiac-specificdeletion of the Ras GTPase-activating protein neurofibromin thatinhibits Ras signaling (Rose 2010, Xu 2009). Conversely, transgenicexpression of dominant-negative Raf1 inhibited the hypertrophy due topressure overload.

Recently, investigators have shown that cardiac myocyte-specificknockout of all 4 ERK1/2 alleles resulted in a severe, fatal dilatedcardiomyopathy without increased myocyte death (Kehat 2011). ERK1/2-nullmyocytes were longer and narrower than those from control animals.PTPN11 (Shp2) knockout that decreased ERK1/2 activation also resulted inan elongated myocyte morphology and dilated cardiomyopathy (Kontaridis2008).

Conversely, myocytes from constitutively active MEK1 transgenic micewere shorter and wider (Kehat 2011). In contrast to the ERK1/2 and Shp2knockout mice, the inventors found that deletion of the down-streameffector RSK3 resulted in a milder phenotype, with the defect inconcentric growth significant only after TAC and Iso infusion. Together,these observations are consistent with the hypothesis that ERK1/2signaling through RSK3 promotes stress-induced concentric growth ofcardiac myocytes independently of other signaling pathways that regulateeccentric hypertrophy.

The inventors found that RSK3 was activated in myocytes by not onlyERK1/2 but also ERK5. There is evidence that MEK5-ERK5 signalingprimarily induces eccentric myocyte hypertrophy (Nicol 2001), althoughERK5 also may contribute to concentric growth (Kimura 2010).

The data obtained using the RSK knockout mouse establish a function forRSK3 in pathological remodeling. Without being bound by any particulartheory, it is also possible that RSK3 has a role in physiologichypertrophy. For example, the myocytes isolated from unstressedRSK3^(−/−) mice tended to be smaller in both width and length (FIGS. 9and 29 of U.S. Pat. No. 9,132,174). In addition, after swimming,RSK3^(−/−) biventricular weight was less than that of wild-type mice,albeit not significantly after normalization by body weight (FIG. 18 ofU.S. Pat. No. 9,132,174).

It is remarkable that even though RSK3 constitutes a minority of thetotal RSK in the myocyte (FIGS. 24 and 30 of U.S. Pat. No. 9,132,174),RSK3 activity is, nevertheless, required for myocyte growth. Thedifferential anchoring of RSK3 by scaffold proteins provides a mechanismby which RSK3 may specifically function in vivo. Scaffolds are likely tobe most important for enzymes such as RSK3 that are low in abundance andthat have broad intrinsic substrate specificity. RSK protein kinasescatalyze the phosphorylation of RxRxx(S/T) sites and overlap inspecificity with other AGC kinases (Anjum 2008). By co-localizingenzymes, their upstream activators, and substrate effectors, scaffoldscan accelerate the kinetics of signaling, amplify responses, increasespecificity in enzyme catalysis, and direct signaling to specificsubcellular compartments (Good 2011). The prior art provides limitedguidance with respect to RSK compartmentation in cells or participationin multi-molecular signaling complexes. On mitogen stimulation,cytosolic RSK1 (and potentially other RSK isoenzymes) can transientlytranslocate to the plasma membrane, whereas activated RSK tends to beenriched in the nucleus (Anjum 2008). In neurons, RSKs bind PDZdomain-containing proteins via their conserved C-terminal STxL peptides,directing the kinases to substrates involved in synaptic transmission(Thomas 2005). By another mechanism, RSK1 binds type 1 protein kinase Aand D-AKAP-1, a mitochondrion-localized scaffold (Chaturvedi 2006, Huang1997). Consistent with the fact that the inventors can only detect RSK3in myocytes after immunoprecipitation, the inventors have not been ableto detect endogenous RSK3 protein by immunocytochemistry. Whenoverexpressed at a low level, HA-RSK3 was enriched at the nuclearenvelope, the predominant location for mAKAPβ in the cardiac myocyte(Pare 2005). By characterizing in detail the protein-protein interactionbetween the unique RSK3 N terminus and mAKAPβ, the inventors haveidentified a new mechanism by which RSK3 can be specifically anchored by≥1 scaffolds that may be targeted to different signaling compartments.

The inventors demonstrated the functional significance of this RSK3anchoring using a competing binding peptide (mAKAP RBD) that inhibitedmyocyte hypertrophy.

The regulation of NHE1 by RSK1/2 has spurred recent interest in usingRSK inhibitors to treat heart disease (Avkiram 2008). The inventors showthat RSK3 knockout reduced TAC-induced hypertrophy without diminishingcardiac function and while inhibiting the expression of genetic markersfor pathological remodeling. RSK inhibition may have multipleapplications, including its use in acquired diseases such ashypertension (pressure overload) and for the treatment of theaforementioned Rasopathies. Recently, a Noonan syndrome mouse model(Raf1L⁶¹³V knock-in) mouse was treated with PD0325901, resulting in theattenuated progression of cardiac hypertrophy cardiomyopathy and otherNoonan characteristics (Wu 2011). Targeting of RSK3 offers analternative approach to avoid some of the harmful side effects of globalERK pathway inhibition. The use of RSK3 inhibitors that eithercompetitively bind the active site or disrupt anchoring are offered asnovel cardiac therapies.

Example 2

Remodeling of the extracellular matrix and the induction of myocardialinterstitial fibrosis is an important factor contributing to thedevelopment of heart failure in cardiac disease (Spinale 2013, Edgley2012). Increased deposition of fibrillar collagen and disruption of thenormal cellular architecture of the myocardium can result in decreasedcompliance and both diastolic and systolic dysfunction, as well asarrhythmia due to interference with the electrical conduction system.p90 ribosomal S6 kinases (RSK) are pleiotropic protein kinases that areactivated in myocytes in response to many stress-related stimuli (Anjum2008, Sadoshima 2005, Kodama 2000). The inventors have shown that type 3RSK (RSK3) is required for the induction of concentric myocytehypertrophy in mice subjected to pressure overload (Li 2013). Activatedby sequential phosphorylation by extracellular signal-regulated kinases(ERKs) and 3′-phosphoinositide-dependent kinase 1, RSK3 is one of fourRSK family members expressed in the heart (Anjum 2008). Remarkably, eventhough RSK3 comprises a minority of RSK enzyme in cardiac myocytes, RSK3is required for hypertrophy (Li 2013). Due to its role in pathologicalhypertrophy, the inventors have suggested that RSK3 targeting might bebeneficial in the prevention of heart failure. To our knowledge,however, the prior art is deficient in showing whether RSK familymembers also regulate cardiac fibrosis. In this study, the inventors nowshow a role for RSK3 in interstitial fibrosis that is independent of itsfunction in hypertrophic signal transduction.

Hypertrophic cardiomyopathy (HCM) is the most commonly inherited heartdefect (1 in 500 individuals) and the leading cause of sudden death inchildren, accounting for 36% of sudden deaths in young athletes (Maron2013). HCM is caused by dominant mutations in sarcomeric proteins thattypically induce myocyte hypertrophy and disarray and interstitialfibrosis. However, the phenotype and clinical course resulting from HCMmutations can vary such that genotype-positive patients without leftventricular hypertrophy can display myocardial fibrosis, diastolicdysfunction, and ECG abnormalities (Maron 2013). Studies usingtransgenic mice also indicate that the phenotype of HCM mutationsdepends upon genetic background (Prabhakar 2001, Michele 2002). Asdescribed below, expression of the HCM mutation Glu180Gly amino acidsubstitution of the thin filament protein α-tropomyosin (TM180) in miceof a mixed C57BL/6; FVB/N background results in a small left ventriclewith interstitial fibrosis. The inventors show that RSK3 is required inthis non-hypertrophic HCM model for the development of interstitialfibrosis and the signs of left-sided heart failure.

Methods

Supplemental Material

Reagents: Primary antibodies included mouse 1F6 monoclonal anti-RSK3(Abnova, cat #H00006196-M01) that detects all RSK family members (Li2013), OR43 rabbit anti RSK3, and N-16 goat anti-RSK3 (Santa CruzBiotechnology). Secondary antibodies included horseradish peroxidase(HRP)-conjugated donkey secondary antibodies (Jackson ImmunoResearch).RSK3 immunoprecipitation was performed as previously described (Li2013).

Mice: All experiments involving animals were approved by theInstitutional Animal Care and Use Committee at the University of Miami.The RSK3^(−/−) C57BL/6 mouse was mated to the TM180 transgenic FVB/Nmouse (Li 2013, Prahakar 2001). All mice studied were littermates fromRSK3^(−/+) X TM180; RSK3^(−/+) breedings, such that the backgroundstrain was 50:50 C57BL/6; FVB/N. All four genotypes were present intypical Mendelian proportion. Unless otherwise specifies, allexperiments were performed with mice that were 16 weeks of age.Genotyping was performed at weaning by PCR using tail biopsy samples aspreviously described (Li 2013, Prahakar 2001). Echocardiography: A Vevo770TM High-Resolution In Vivo Imaging System (VisualSonics) with a RMVTM707B “High Frame” Scan-head was used for imaging. Mice were anesthetizedwith 1.5% isoflurane for both B-mode and M-mode imaging.

Histochemistry: Heart tissue was fixed in 3.7% formaldehyde.De-paraffinized 5 μm tissue sections were stained using the PicrosiriusRed Stain Kit (Polysciences) and Alexa Fluor 555 Wheat Germ Agglutininconjugate (Invitrogen) as recommended by the manufacturers. Thecross-section area of >150 myocytes in >3 distinct regions of the leftventricle were measured per heart using the wheat germ agglutininsections. Collagen content was assayed using the Picrosirius Red stainedsections and linearly polarized light microscopy for >3 4× objectivefields per heart. Note that while linearly polarized light microscopy isa highly specific assay for fibrillar collagen, the values obtained arean underestimate of total collagen content. TUNEL staining for bothfixed cells and tissue sections was performed using the In Situ CellDeath Detection Kit, TMR red (Roche). Morphometrics and collagen contentwere measured using IPLab microscope software (BD Biosciences). Allanalyses were performed by a blinded investigator.

RNA Assay: Total RNA was quantified with a Nanodrop 8000Spectrophotometer (ThermoScientific) and quality controlled using with aBioanalyzer 2100 and the RNA 6000 Nano kit(Agilent). The NanoStringassay is based on direct, multiplexed measurement of gene expressionwithout amplification, utilizing fluorescent molecular barcodes andsingle molecule imaging to identify and count multiple transcripts in asingle reaction. Briefly, 100 ng total RNA were hybridized in solutionto a target-specific codeset overnight at 65° C. The codeset containeddual, adjacently placed 50 bp oligonucleotide probes against the entirepanel of genes, one set of probes fluorescently bar-coded and the otherbiotinylated. The hybridization reactions were loaded onto theNanoString Prep station which removes excess oligonucleotides and bindsthe hybridized mRNA to the Streptavidin-coated cartridge surface. Thecartridges were loaded onto the NanoString Digital Analyzer, and 1150fields of view were fluorescently scanned to count only those individualmRNAs bound to both a biotinylated and fluorescently bar-coded probe.Datasets for each RNA sample were normalized to internal positivecontrols and background-subtracted. Probe sequences are available uponrequest.

Statistics: For all experiments, n refers to the number of individualmice. All data are expressed as mean±s.e.m. p-values were calculatedusing two-tailed Student's t-tests, paired or un-paired as appropriate,and are not corrected for multiple comparisons. Repeated symbolsrepresent p-values of different orders of magnitude: * p<0.05, **p<0.005, *** p<0.0005.

Results

FVB/N TM180 transgenic mice were crossed with C57BL/6 RSK3 knock-outmice such that all mice were of a mixed 50:50 background. RSK3expression was slightly higher (˜25%, p=0.12) in the TM180 mice, whileabsent in RSK3^(−/−) mice, with no evidence of compensatory changes inthe expression of other RSK family members (FIG. 32 of U.S. Pat. No.9,132,174). Expression of the TM180 transgene was evident by theexpected change in α-tropomyosin bands detected by total protein stain(Prabhakar 2001). In these mice of mixed lineage, the TM180 transgeneinduced a small heart phenotype that included a reduced biventricularweight (21%) and left ventricular myocytes with a proportionally smallercross-section area (FIGS. 32 and 34 of U.S. Pat. No. 9,132,174). Byechocardiography, the TM180 mice had reduced left ventricular internaldimensions, but increased contractility, i.e., both increased fractionalshortening on M-mode and increased endocardial fractional areashortening on B-mode (FIGS. 31 and 36 of U.S. Pat. No. 9,132,174). Thatthe changes in the TM180 left ventricle were pathologically importantwere implied by both an increased atrial weight and a significant,albeit small (12%) increase in wet lung weight, consistent with thepresence of mild pulmonary edema and left-sided heart failure (FIGS. 32Dand 34 of U.S. Pat. No. 9,132,174).

RSK3 knock-out had little effect on the heart in the absence of theTM180 transgene. While RSK3 knock-out did not reverse the small heartphenotype of the TM180 mouse nor prevent the atrial enlargement (FIGS.32B,C and FIG. 34 of U.S. Pat. No. 9,132,174), the cardiac function ofTM180; RSK3^(−/−) mice was more like wildtype mice, including a 29%lesser decrease in short axis dimension by echocardiography (FIGS. 31and 36 of U.S. Pat. No. 9,132,174). Notably, the increase in fractionalshortening and endocardial fractional area shortening due to the TM180transgene were both attenuated by ˜50% by RSK knock-out. That the more“normal” cardiac function of the TM180; RSK3^(−/−) mice wasphysiologically important was implied by the observation that wet lungweight was no longer increased following RSK3 knock-out (FIG. 32D andFIG. 34 of U.S. Pat. No. 9,132,174).

There was no increase in cellular death associated with the TM180transgene at 16 weeks of age (˜10⁻⁴ TUNEL-positive nuclei for allcohorts, data not shown). However, trichrome staining of the TM180hearts revealed a patchy interstitial fibrosis in the myocardium notpresent in wildtype mice that was greatly reduced in the absence of RSK3(FIG. 33A,B of U.S. Pat. No. 9,132,174). Likewise, picrosirius redstaining showed that fibrillar collagen content was increased by theTM180 transgene only in the presence of RSK3 (FIG. 33C of U.S. Pat. No.9,132,174). These results were corroborated by assay of the expressionof genes involved in cardiac function and remodeling (FIG. 35 of U.S.Pat. No. 9,132,174). Notably, genes involved in cardiac fibrosis,including Col8a1 and Postn, (Oka 2007) encoding collagen type α1 andperiostin, respectively, were induced by the TM180 transgene in aRSK3-dependent manner (FIG. 33D,E of U.S. Pat. No. 9,132,174).

Discussion

When expressed in FVB/N mice, the HCM TM180 mutation results inconcentric left ventricular hypertrophy, extensive fibrosis, atrialenlargement, and death within 5 months (Prabhakar 2001). In contrast,expression of the TM180 mutation in C57BL/6 mice resulted in noventricular hypertrophy or fibrosis and a lower heart weight (Michele2002). The inventors found that in a mixed C57BL/6; FVB/N background,the TM180 transgene induced an intermediate phenotype, includingdecreased ventricular and increased atrial weights, smaller ventricularmyocytes, interstitial fibrosis, and increased contractility byechocardiography. The TM180 mutation is thought to induce cardiomyopathyas a result of the increased Ca²⁺ sensitivity and increased maximumtension generation of TM180 filaments (Prabhakar 2001). Hence,increasing Ca²⁺ reuptake through manipulation of phospholamban and thesarco/endoplasmic reticulum Ca²⁺-ATPase 2A (SERCA2A) can ameliorate theTM180 FVB/N phenotype (Gaffin 2011). The inventors have utilized theTM180 transgenic mouse to investigate the role of RSK3 in cardiacfibrosis. While the inventors and others have found that total heartERK1/2 is activated in TM180 FVB/N mice (Gaffin 2011), in the mixedbackground mice, total ERK1/2 and RSK phosphorylation was not increased.Instead, the inventors only noted a slight increase in RSK3 proteinlevels (FIG. 32A of U.S. Pat. No. 9,132,174). Importantly, RSK3knock-out blocked the TM180 associated induction of fibrotic geneexpression and interstitial fibrosis, as well as improving cardiacfunction both in terms of echocardiographic findings and wet lungweight. These findings complement our previous observation that RSK3 isspecifically required for pathological cardiac hypertrophy. Withoutbeing bound by a particular theory, the inventors suggest that due toRSK3 anchoring through its unique N-terminal domain to scaffold proteinssuch as mAKAP (muscle A—kinase anchoring protein), RSK3 serves a uniquefunction in the heart, despite the higher level of expression of otherRSK isoenzymes (Li 2013). There are reports that RSK phosphorylation ofCEBP/β is involved in pathological fibrosis of the liver and lung, butthere is no published data relating to RSK family members in the heart.The inventors suggest that specific RSK3 inhibition should now beconsidered more broadly as a therapeutic target both in hypertrophic andfibrotic heart diseases.

Example 3

mAKAP gene structure and the strategy for a conditional mAKAP allele.The mAKAP gene contains 12 common (light blue) and 3alternatively-spliced exons (beige and yellow). A targeting vectorcontaining negative (tk) and positive (neo) selectable markers wasdesigned to conditionally delete the common Exon 9. The inventorsobtained 6 targeted ES cell clones as shown by Southern blots. Afterbreeding of targeted mice, the neo cassette was deleted by mating to aFLP recombinase transgenic. Mating to a mouse expressing cre recombinasewill result in the deletion of Exon 9 (KO allele), producing a frameshift and introduction of a stop codon (red) in Exon 10. Mousegenotyping is being performed by PCR of genomic DNA with primers 44 and45. (FIG. 39 of U.S. Pat. No. 9,132,174). For the western blot: mAKAPEx9^(fl/fl); Tg(Myh6-cre/Esr1) mice (lanes 2 and 3) and mAKAPEx9^(fl/fl) (lane 1) and Tg(Myh6-cre/Esr1) (lane 4) control mice werefed 500 mg tamoxifen/kg dry food for one week before the hearts werecollected to prepare total RNA and protein extracts. RT-PCR wasperformed using primers located within mAKAP exons 4 and 11 which yielda 1022 bp for wildtype (and floxed) mRNA and a 901 bp product for amAKAP mRNA species lacking exon 9. While control β-actin mRNA wassimilarly detected for all samples (bottom panel), >90% less PCR productwas obtained for CKO mouse hearts (top panel, lanes 2 and 3) compared tothat observed with the control hearts (lanes 1 and 4). Western blotswere performed using VO54 mAKAP-specific antibody. No mAKAP protein wasdetectable for heart extracts prepared from CKO mice (lanes 2 and 3, toppanel). Equal loading was determined by Ponceau total protein stain(bottom panel).

mAKAP Ex9^(fl/fl); Tg(Myh6-cre/Esr1) mice (fl/fl; MCMTg) andTg(Myh6-cre/Esr1) (MCM Tg) at 8 weeks of age were fedtamoxifen-containing chow for one week, rested for one week and thensubjected for to 2 weeks of Transverse Aortic Constriction beforeanalysis (FIG. 40 of U.S. Pat. No. 9,132,174). mAKAP gene deletioninhibited the development of concentric hypertrophy in response topressure overload. Subsequent studies showed that mAKAP gene deletionprevented the development of heart failure in response to chronicpressure overload, inhbiting cardiac remodeling and providing a survivalbenefit (Kritzer 2014).

Transverse Aortic Constriction: All tools were sterilized with aGerminator 500 Dry Sterilizer and Betadine Solution (10% povidone-iodinetopical solution). Anesthesia was induced with 5% isoflurane andmaintained with 2% isoflurane and 100% oxygen at a flow rate of 1.5L/min using a SurgiVet flow regulator via nose cone. Loss ofconsciousness was verified by toe pinch. Mouse fur over the left chestand sternum was removed with a calcium hydroxide lotion (e.g. Nair), andthe surgical site was sterilized with betadine. The skin was incisedexposing the pectoralis muscle and the second left intercostal space.The pectoralis muscle and the second rib were blunt dissected andretracted revealing the thymus within the mediastinum. The lobes of thethymus were retracted to reveal the transverse aortic arch as well asthe right innominate and left common carotid arteries. Blunt dissectionthough the connective tissue between these two arteries and under theaorta allowed for the passage of a 6-0 silk using a modified ligationaid (Fine Science Tools 18062-12). A 27 gauge needle was placed on topof the aorta and the 6-0 silk was tied around the needle. The needle wasremoved, leaving a constricted aorta. The chest was closed in two layerswith 5-0 Polysorb Suture. Isoflurane administration was terminated, andthe mice were maintained on 100% oxygen by nose cone until conscious.Immediately post-operatively, buprenorphrine (0.05-0.1 mg/kg s.c.) wasadministered and then q12 h prn. The mice were allowed to recover undera heat lamp until alert and active. Sham-operated mice that experienceall but the placement of the aortic ligature served as controls.

Echocardiography: Mice minimally anesthetized with 1-2% isoflurane werestudied using a Vevo 770®, High-Resolution Imaging System(VisualSonics). The pressure gradient following TAC was calculated fromthe pulse wave Doppler velocity at the point of ligation as follows:P=4v²; P=the induced pressure gradient (in mmHg) and v=the velocityacross the constriction (in m/s). (FIG. 40 of U.S. Pat. No. 9,132,174).

Example 4

RSK3 anchoring is important for rat ventricular myocyte hypertrophy.

FIG. 8A of U.S. Pat. No. 9,132,174 shows mAKAPβ complexes wereimmunoprecipitated using FL100 mAKAP antiserum from PE-treated,adenovirus-infected neonatal rat ventricular myocytes expressing myc-GFPor myc-GFP-RBD (mAKAP 1694-1833) and detected with the pan-RSK 1F6 andmAKAP 211 antibodies. B. Transfected myocytes expressing GFP or GFP-RBD(green) were stained with α-actinin (blue) and ANF (red) antibodies.Bar=20 μm. C. Cross-section area of myocytes. n=5. D. Fraction ofmyocytes expressing ANF. n=3. * p-values comparing to GFP-expressingsamples. † p-values comparing to no agonist control.

Neonatal rat myocytes isolation and culture: 1-3 day old Sprague-Dawleyrats were decapitated and the excised hearts placed in 1×ADS Buffer (116mmol/L NaCl, 20 mmol/L HEPES, 1 mmol/L NaH₂PO₄, 5.5 mmol/L glucose, 5.4mmol/L KCl, 0.8 mmol/L MgSO₄, pH 7.35). The atria were carefully removedand the blood washed away. The ventricles were minced and incubated with15 mL 1×ADS Buffer containing 3.3 mg type II collagenase (Worthington,230 U/mg) and 9 mg Pancreatin (Sigma) at 37° C. while shaking at 80 RPM.After 15 minutes, the dissociated cardiac myocytes were separated bycentrifugation at 50×g for 1 minute, resuspended in 4 mL horse serum andincubated 37° C. with occasional agitation. The steps for enzymaticdigestion and isolation of myocytes were repeated 10-12 times tomaximize yield. The myocytes were pooled and spun down again at 50×g for2 minutes and resuspended in Maintenance Medium (DMEM:M199, 4:1)supplemented with 10% horse serum and 5% fetal bovine serum. To removeany contaminating fibroblasts, the cells were pre-plated for 1 hourbefore plating on gelatin-coated tissue culture plasticware. Thisprocedure yields >90% pure cardiac myocytes. After 1 day in culture, themedia was changed to maintenance medium containing 0.1 mmol/Lbromodeoxyuridine to suppress fibroblast growth.

Experiments were initiated 1 day after myocyte isolation. Adenoviralinfection was performed by addition of adenovirus (multiplicity ofinfection=5-50) to the media. Plasmids and siRNA oligonucleotides weretransfected using Transfast (Promega) and Dharmafect (Thermofisher),respectively, as recommended by the manufacturers using cells culturedin maintenance medium supplemented with 4% horse serum. Starting the dayafter gene transduction, the cells were treated for as long as 2 days,as indicated for each experiment.

Immunocytochemistry: Cultured neonatal cardiomyocytes on plasticcoverslips were fixed in 3.7% formaldehyde in PBS, permeabilized with0.3% Triton X-100 in PBS, and blocked with PBS containing 0.2% BSA and1% horse serum for 1 hour. The slides were then sequentially incubatedfor 1 hour with primary and Alexa fluorescent dye-conjugatedspecific-secondary antibodies (Invitrogen, 1:1000) diluted in blockingbuffer. The slips were washed three times with blocking buffer. 1 μg/mLHoechst 33258 was included in the last wash stop to label nuclei. Slideswere sealed in SlowFade Gold antifade buffer (Invitrogen) forfluorescent microscopy. Wide-field images were acquired using a LeicaDMI 6000 Microscope.

AAV9 containing the indicated CIS plasmid encoding myc-GFP-mAKAP1694-1833 were injected into neonatal wildtype mice. 2 week TAC wasperformed and analyzed as above at 8 weeks of age. (FIGS. 41-42 of U.S.Pat. No. 9,132,174). Expression of myc-GFP-mAKAP 1694-1833 inhibitedconcentric hypertrophy in vivo in the adult heart.

Example 5

The present invention provides a novel biologic agent for mAKAP RNAi inthe cardiac myocyte in vivo useful in human patients.

mAKAPβ signalosomes have been studied using a variety of technologies,including (1) the expression of anchoring disruptor peptides thatinhibit signaling enzyme association with the scaffold (Kapiloff 2009,Dodge-Kafka 2010, Vargas 2012, Li 2013, Li 2013), (2) by mAKAP (AKAP6)gene knock-out (Kritzer 2014), and (3) by RNA interference (RNAi) ofmAKAP expression using both small interfering RNA oligonucleotides(siRNA) or viral and plasmid vector-expressed small hairpin RNA (shRNA)(Pare 2005, Wong 2008).

A sequence previously disclosed that was targeted by siRNA and shRNA formAKAP is rat specific and not present in the human gene:

Sequence NCBI gene Initial Publication GACGAACCTTCCTTCCGAANCBI Reference Sequence: (Pare 2005) (SEQ ID NO: 10)XM_017594342.1 rat AKAP6 base pairs 7483-7501

In order to generate a biologic that would target mAKAP expression inhumans, three 19 base pair sequences were chosen from NCBI referencesequence XM_017021808.1 for human AKAP6 transcript variant X1 mRNA (FIG.8):

Position in Reference Seq # Sequence Sequence (base pairs) 1GGTTGAAGCTTTGAAGAAA (SEQ ID NO: 7) 3094-3112 2GCTAAGAGATACAGAGCTT (SEQ ID NO: 8) 3316-3334 3GGAGGAAATAGCAAGGTTA (SEQ ID NO: 9) 7807-7825

These sequences were incorporated into novel, custom-designed shuttleplasmids for self-complementary adeno-associated virus (AAV) that wouldconfer cardiac myocyte-specific shRNA expression in vivo. The“pscA-TnT-mAKAP shRNA” plasmids were designed as follows (FIGS. 9 and10):

-   -   1) A cDNA cassette was generated using human mir-30a sequences        to confer efficient shRNA expression. The construct was similar,        but not identical to a previously published cassette (Silva        2005): 128 bp of human mir30a 5′ sequence with 2 mismatches—2 bp        (CG)—AKAP6 sequence in sense orientation—19 bp loop        sequence—AKAP6 sequence in antisense orientation—2 bp (CT)—130        bp of human mir30 sequence with 1 mismatch.    -   2) The cDNA cassette was located 3′ to a fragment of the chicken        cardiac troponin T promoter that confers cardiac myocyte        specific expression (Prasad 2011) and 5′ to a SV40-derived        polyadenylation sequence (SV40 genome bp 2599-2769).    -   3) The entire shRNA minigene was flanked by AAV2 ITR sequence        (NC_001401.2 bp 4489-4664 in antisense orientation on the 5′ end        and bp 4559-4662 on the 3′ end) in order to direct production of        a self-complementary AAV (scAAV) biologic (Wang 2003).

Testing in cultured rat neonatal rat ventricular myocytes revealed thattransfection with these plasmids resulted in decreased expression ofmAKAPβ to varying degrees, with the plasmid for target #3 being mosteffective at removing mAKAPβ (data not shown). Therefore, pscA-TnT-mAKAPshRNA #3 was used to generate scAAV particles with the cardiotropicserotype 9 capsid protein for in vivo experimentation. The scAAV weregenerated by the University of Pennsylvania Vector Core with supportprovided by the National Heart, Lung, and Blood Institute Gene TherapyResource Program. The scAAV virus were tested by tail vein injectioninto adult mice. Three weeks after injection, hearts were collected andanalyzed by western blot. mAKAPβ expression was repressed >90% by asingle intravenous (IV) dose of 5×10¹¹ viral genomes (vg) of scAAV-mAKAPshRNA #3 when compared to mice injected with a scAAV control shRNA virusor non-injected controls (FIG. 11).

Example 6

Coronary heart disease is a leading cause of heart failure (WritingGroup 2016), and an ability to block heart failure in ischemic diseaseby single dose scAAV-mAKAP shRNA is desirable. In mice, left coronaryartery ligation results in a large, scarred transmural infarct (sparingthe ventricular septum) accompanied by ventricular dilatation andremodeling of the remaining myocardium (Kumar 2005). 8 weeks post-MI,control mice are expected to be in heart failure with low ejectionfraction and cardiac output (systolic dysfunction), atrial hypertrophy,and pulmonary edema (increased wet lung weight).

8-week old C57BL/6 WT mice were subjected to permanent left coronaryartery ligation or sham thoracotomy and studied by serialechocardiography until euthanized 8 weeks post-survival surgery (FIG.12A), using the following methods:

Method for Ligation of the Left Coronary Artery: Mice were anesthetizedwith 5% isoflurane for induction and then 2.5-3% for maintenance.Orotracheal intubation was performed using a 16G catheter, and the mousewas then ventilated mechanically using a minivent ventilator. The skinover the site of left lateral thoracotomy was prepped and draped insterile fashion using providone-iodine 10% solution. A heating pad wasused to keep mice warm during procedures to prevent heat loss.Surgically sterile non-medicated ophthalmic ointment was applied to theeyes preoperatively to prevent corneal drying. Survival surgery wasperformed under microscope view. Once adequate sedation was achieved,the chest was opened via left lateral thoracotomy at the fourthintercostal space. If muscle bleeding was present, hemostasis wasachieved by the using a thermal cauterizer (e.g. fine tip Bovie). A 3 mmretractor was used to separate the ribs. Following pericardiotomy, theleft coronary artery was ligated with a 8-0 prolene suture to produce ananterior MI. The chest was closed in 3 layers with 5-0 absorbable suture(muscle), 7-0 (for 2 ligatures in the ribs) and 6-0 for the skin.Buprenorphine slow release (Bup-SR-LAB) 0.5-1 mg/kg s.c. wasadministered in a single dose immediately after surgery to control painfor 72 hr. Fluid replacement was also administered as needed immediatelyafter surgery (e.g., sterile saline solution 0.9%, IP). The mice wereallowed to recover until alert and active. Sham-operated mice thatexperience all but the placement of the coronary artery ligature serveas controls.

Echocardiography: Mice minimally anesthetized with 1-2% isoflurane werestudied using a Vevo 2100®, High-Resolution Imaging System(VisualSonics). B- and M-mode images were obtained for mice underanesthesia at various time-points. Posterior wall and anterior walldiastolic and systolic thicknesses and left ventricular cavityend-diastolic (LVEDD) and end-systolic diameters (LVESD) were measured,permitting estimation of LV volumes, fractional shortening and ejectionfraction.

Upon confirmation of reduced ejection fraction (EF ≤40%) 2 days afterLigation of the Left Coronary Artery survival surgery (MI cohorts), micewere randomized and injected the following day with 5×10¹¹ vg I.V. ofeither scAAV-mAKAP-shRNA #3 or scAAV-control-shRNA. The average EF forthe MI cohorts before treatment was 31.66% and 32.79% for mAKAP andcontrol shRNA cohorts, respectively. After scAAV treatment, cohorts wereas follows: mAKAP-shRNA-MI (n=10), control-shRNA-MI (n=6),mAKAP-shRNA-Sham (n=4), control-shRNA-Sham (n=5). Administration ofmAKAP-shRNA post-MI resulted in amelioration of systolic dysfunction asevidenced by normalization of EF from day 2 to weeks 2, 4 and 8 post-MI,while control-shRNA-MI animals displayed a progressive deterioration ofEF after ischemic injury (FIG. 12B). At 8 weeks post MI, EF inmAKAP-shRNA-MI was not significant different from the mAKAP- andcontrol-shRNA-Sham animals (44.3±3.3% vs. 54.6±1.7% & 54.5±2.0%), whileit was higher than in control-shRNA-MI animals (18.0±4.3%, P<0.0001). Inaddition, 8 weeks after coronary artery ligation, mAKAP-shRNA-MI micedisplayed thicker left ventricular (LV) anterior wall in systole(0.9±0.1 mm vs. 0.5±0.1 mm for control-shRNA-MI, P<0.05) and smaller LVend-systolic-volumes, (72.1±8.6 μL vs. 137.3±26.3 μL forcontrol-shRNA-MI; P<0.01) than control-shRNA-MI mice. Consequently,post-mortem analysis of pulmonary edema showed that treatment withscAAV-mAKAP-shRNA #3 attenuated the development of heart failure (FIG.13), such that wet lung weight for mAKAP-shRNA-MI mice was significantlyless than for the control-shRNA-MI cohort (7.7±0.2 vs. 10.6±1.2 mg/mmtibial length, P<0.01).

These results show that inhibition of mAKAPβ expression using the newscAAV-mAKAP shRNA #3 biologic drug as a treatment after myocardialinfarction ameliorates cardiac dysfunction, preserving cardiac structureand preventing the development of heart failure in mice.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference and full citations can be found in U.S. Pat.No. 9,132,174. While this invention has been particularly shown anddescribed with references to preferred embodiments thereof, it will beunderstood by those skilled in the art that various changes in form anddetails may be made therein without departing from the scope of theinvention encompassed by the appended claims.

What is claimed is:
 1. A composition comprising a vector encoding asmall hairpin ribonucleic acid (“shRNA”) against human mAKAP, andwherein the small hairpin ribonucleic acid comprises the followingnucleotide sequence: GGAGGAAATAGCAAGGTTA (SEQ ID NO: 9).
 2. Apharmaceutical composition comprising an amount of the composition ofclaim 1 which is therapeutically effective when administered to apatient in need thereof to protect the patient from heart damage.
 3. Thecomposition of claim 1, wherein the vector is a viral vector.
 4. Thecomposition of claim 3, wherein the viral vector is an adeno-associatedvirus vector (AAV).
 5. The composition of claim 3, wherein the viralvector comprises human microRNA (miR)-30a sequences.
 6. The compositionof claim 3, wherein the viral vector comprises a shRNA minigene, whereinthe minigene is flanked by a complete AAV2 inverted terminal repeat(ITR) on the 5′ end and a deleted AAV2 ITR on the 3′ end.
 7. Thecomposition of claim 3, wherein the viral vector comprises a chickencardiac troponin T promoter.
 8. The composition of claim 3, wherein theviral vector comprises a SV40 polyadenylation site.
 9. The compositionof claim 4, wherein the AAV is an AAV serotype
 9. 10. A method ofinhibiting the expression of human mAKAPβ, comprising contacting mRNAencoding mAKAPβ with the composition of claim
 1. 11. A method ofprotecting the heart from damage, comprising administering to a patientat risk of such damage, a pharmaceutically effective amount of thecomposition of claim
 1. 12. A method of treating or preventing heartdisease, comprising administering to a patient in need thereof, apharmaceutically effective amount of the composition of claim
 1. 13. Amethod of treating or preventing myocardial infarction, comprisingadministering to a patient at risk of such damage, a pharmaceuticallyeffective amount of the composition of claim 1.